U.S. patent application number 15/513102 was filed with the patent office on 2017-09-21 for polymeric bile acid derivatives inhibit hepatitis b and d virus and ntcp transport.
This patent application is currently assigned to Huahui Health Ltd.. The applicant listed for this patent is Huahui Health Ltd.. Invention is credited to Wenhui Li, Yang Liu, Bo Peng, Xiangbing Qi, Zhiqiang Wang, Huan Yan, Lei Zhang.
Application Number | 20170266206 15/513102 |
Document ID | / |
Family ID | 55580345 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170266206 |
Kind Code |
A1 |
Li; Wenhui ; et al. |
September 21, 2017 |
POLYMERIC BILE ACID DERIVATIVES INHIBIT HEPATITIS B AND D VIRUS AND
NTCP TRANSPORT
Abstract
The invention provides for treating HBV or HDV infection or
inhibiting human sodium taurocholate co-transporting polypeptide
(hNTCP) with a polymeric bile acid or salt thereof, and
pharmaceutical compositions comprising a polymeric bile acid or
salt thereof, and a second HBV or HDV medicament.
Inventors: |
Li; Wenhui; (Beijing,
CN) ; Qi; Xiangbing; (Beijing, CN) ; Yan;
Huan; (Beijing, CN) ; Liu; Yang; (Beijing,
CN) ; Wang; Zhiqiang; (Beijing, CN) ; Peng;
Bo; (Beijing, CN) ; Zhang; Lei; (Beijing,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Huahui Health Ltd. |
Beijing |
|
CN |
|
|
Assignee: |
Huahui Health Ltd.
Beijing
CN
|
Family ID: |
55580345 |
Appl. No.: |
15/513102 |
Filed: |
September 28, 2015 |
PCT Filed: |
September 28, 2015 |
PCT NO: |
PCT/CN2015/091000 |
371 Date: |
March 21, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/585 20130101;
A61K 31/58 20130101; C07J 41/0055 20130101; A61P 31/20 20180101;
A61K 31/575 20130101; C07J 9/005 20130101; C07J 43/003 20130101;
C07J 41/0061 20130101; A61P 31/14 20180101; C07J 9/00 20130101;
C07J 71/0047 20130101; A61P 43/00 20180101; C07J 41/005 20130101;
A61K 45/06 20130101; A61K 31/575 20130101; A61K 2300/00 20130101;
A61K 31/585 20130101; A61K 2300/00 20130101; A61K 31/58 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 31/585 20060101
A61K031/585; A61K 31/58 20060101 A61K031/58; A61K 45/06 20060101
A61K045/06; A61K 31/575 20060101 A61K031/575 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 2014 |
CN |
PCT/CN2014/087655 |
Claims
1.-16. (canceled)
17. A bile acid monomer, having a structure independently of
formula I: ##STR00227## wherein each C1-C24 is independently,
optionally substituted: with a hydrocarbon or heterohydrocarbon or
heteroatom-containing functional group, or with an
optionally-substituted alkyl or heteroalkyl, alkenyl or
heteroalkenyl, alkynyl or heteroalkynyl, alkoxyl or heteroalkoxy,
including cyclic and substituted forms of each, aryl and
heteroaryl, wherein hetero-forms comprise 1-4 heteroatoms such as
N, O, P and S, or with a functional group selected from hydroxyl,
haloformyl, carbonyl, aldehyde, carboxyl, ester, acetal,
carboxamide, hydroperoxyl, epoxide, peroxide, oxime, amine, imine,
imide, amide, quaternary ammonium salt, amine oxide, azide, azo,
diazo, azido, aziridine, diaziridine, hydrazine, hydrzone,
aldimine, isocyanide, isocyanate, isothiocyanate, cyanate, nitrate,
nitrile, nitrite, nitride, nitro, nitroso, silane, alkylsilane,
siloxane, halosilane, phosphine, phosphorite, phosphate,
thiophosphonate, quaternary phosphonium salt, phosphono,
phosphides, sulfide, sulfite, sulfonate, thiocyanate, thiosulfate,
sulfoxide, sulfimide, sulfone, sulfoximines, sulfonium, and
sulfhydryl, or with substituted or unsubstituted (C1-C4)alkyl,
(C2-C4)alkenyl, (C6-C8)alkenyl, (C2-C4)alkynyl, (C6-C8)alkynyl,
(C1-C4)alkoxyl, (C6-C8)alkoxyl, 3-oxetanyloxy,
3-tetrahydrofuranyloxy, Cl, F, fluoro-substituted (C1-C2)alkyl,
(C1-C4)alkyl-SO.sub.2--, (C3-C6)cycloalkyl, or a (C5-C6)heterocycle
having 1 or 2 heteroatoms each independently selected from N, O, P,
Si or S.
18. The bile acid monomer of claim 17, wherein each substituent is
independently: OH, OAc (Ac.dbd.COCH.sub.3, Acetate), OBz (Bz=COPh,
benzoyl), OBn (Bn=benzyl), OTs (Ts=tosyl, p-toluenesulfonyl), OMs
(Ms=methanesulfonyl), OSiR.sub.3, OTf
(Tf=trifluoromethanesulfonyl), OTHP (O-tetrahydropyran), OCOR, OR,
NH.sub.2, NHBoc (Boc=tert-butyloxycarbonyl), NHAc
(Ac.dbd.COCH.sub.3, Acetate), NHBz (Bz=COPh, benzoyl), NHTs
(Ts=tosyl, p-toluenesulfonyl), NHTf (Tf=Trifluoromethanesulfonyl),
NHMs (Ms=methanesulfonyl), NHSiR.sub.3, NHR; NBn.sub.2 (Bn=benzyl),
NTf.sub.2 (Tf=trifluoromethanesulfonyl); NHCOR, NR1R2; --N.sub.2,
--N.sub.3; phosphoric acid (--OP(O)(OH).sub.2, sulfuric
acids(--OSO.sub.2OH) or carboxylic acid (--OCOH), carboxylic ester
(--OCOR); phosphorite(--OP(O).sub.mO--) or phosphate
(--OP(OR).sub.mO--), sulfonate, sulfate(--OS(O).sub.mO--), sulfite
(--OS(OR).sub.mO--), disulfite or pyrosulfate, in which m is equal
to 0, 1 and 2; wherein each R, R1 and R2 is independently H, an
alkyl, aryl, alkenyl, alkynyl, carbonyl, or ether radical having up
to 15 carbon atoms, which is branched or unbranched, a cyclo-alkyl,
aryl, alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15
carbon atoms or a phenyl or benzyl radical, which are unsubstituted
or mono-, bis- or trisubstituted by halide, (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine), or an alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether radical having up to 15 carbon
atoms, which is branched or unbranched, a cyclo-(alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15 carbon
atoms, a phenyl or benzyl radical, which are unsubstituted or
mono-, bis-, tri-substituted by halide, (C1-C4)-alkyl,
(C1-C4)-alkoxyl or (C1-C6)-alkylamine), or heteroatom.
19. The bile acid monomer of claim 17, wherein each of C3, C6, C7
and C24 is independently, optionally substituted with: OH, OAc
(Ac.dbd.COCH.sub.3, Acetate), OBz (Bz=COPh, benzoyl), OBn
(Bn=benzyl), OTs (Ts=tosyl, p-toluenesulfonyl), OMs
(Ms=methanesulfonyl), OSiR.sub.3, OTf
(Tf=trifluoromethanesulfonyl), OTHP (O-tetrahydropyran), OCOR, OR,
NH.sub.2, NHBoc (Boc=tert-butyloxycarbonyl), NHAc
(Ac.dbd.COCH.sub.3, Acetate), NHBz (Bz=COPh, benzoyl), NHTs
(Ts=tosyl, p-toluenesulfonyl), NHTf (Tf=Trifluoromethanesulfonyl),
NHMs (Ms=methanesulfonyl), NHSiR.sub.3, NHR; NBn.sub.2 (Bn=benzyl),
NTf.sub.2 (Tf=trifluoromethanesulfonyl); NHCOR, NR1R2; --N.sub.2,
--N.sub.3; phosphoric acid (--OP(O).sub.m(OH).sub.2, sulfuric
acids(--OSO.sub.2OH) or carboxylic acid (--OCOH), carboxylic ester
(--OCOR); phosphorite(--OP(O).sub.mO--) or phosphate
(--OP(OR).sub.mO--), sulfonate, sulfate(--OS(O).sub.mO--), sulfite
(--OS(OR).sub.mO--), disulfite or pyrosulfate, in which m is equal
to 0, 1 and 2; wherein each R, R1 and R2 is independently H, an
alkyl, aryl, alkenyl, alkynyl, carbonyl, or ether radical having up
to 15 carbon atoms, which is branched or unbranched, a cyclo-alkyl,
aryl, alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15
carbon atoms or a phenyl or benzyl radical, which are unsubstituted
or mono-, bis- or trisubstituted by halide, (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine), or an alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether radical having up to 15 carbon
atoms, which is branched or unbranched, a cyclo-(alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15 carbon
atoms, a phenyl or benzyl radical, which are unsubstituted or
mono-, bis-, tri-substituted by halide, (C1-C4)-alkyl,
(C1-C4)-alkoxyl or (C1-C6)-alkylamine), or heteroatom
20. The bile acid monomer of claim 17, wherein C24 is substituted
with: --CH.sub.2OH, --COOM, in which M is an alkali metal, alkaline
earth metal or quaternary ammonium ion; or
--CONHCH.sub.2CH.sub.2SO.sub.3H, --CONHRSO.sub.3H or --COOH,
--COOR, --CONH.sub.2, --CH.sub.2NH.sub.2, --CONH or NRR, wherein
each R is independently H, an alkyl, aryl, alkenyl or alkynyl
radical having up to 15 carbon atoms, which is branched or
unbranched, a cycloalkyl radical having 3 to 15 carbon atoms or a
phenyl or benzyl radical, which are unsubstituted or mono-, bis- or
trisubstituted by halide (e.g. F, Cl, Br), (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine).
21. The bile acid monomer of claim 17, wherein each of C3, C6 and
C7 is independently, optionally substituted with: TABLE-US-00023
--OH, --OAc (Ac = COCH.sub.3, Acetate), --NH2, --NHBoc (Boc = tert-
--OBz (Bz = COPh, benzoyl), BUtyloxycarbonyl), --OBn (Bn = benzyl),
--NHAc (Ac = COCH.sub.3, Acetate), --OTs (Ts = Tosyl,
p-toluenesulfonyl), --NHBz (Bz = COPh, benzoyl), --OMs (Ms =
methanesulfonyl), - --NHTs (Ts = Tosyl, p-toluenesulfonyl), --OR,
--OSiR.sub.3, --NHTf (Tf = Trifluoromethanesulfonyl), --OTr(Tr =
Triphenylmethyl), --NHMs (Ms = methanesulfonyl), --OTf (Tf =
Trifluoromethanesulfonyl), --NBn.sub.2 (Bn = Benzyl) or --OTHP (THP
= tetrahydropyran), --NR1R2, -carbonyl --N.sub.3, -alkyl
wherein each R, R1 and R2 is independently alkyl, aryl, alkenyl or
alkynyl radical having up to 15 carbon atoms, which is branched or
unbranched, a cycloalkyl radical having 3 to 15 carbon atoms or a
phenyl or benzyl radical, which are unsubstituted or mono-, bis- or
trisubstituted by halide (e.g. F, Cl, Br), (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine).
22. A polymeric bile acid comprising 2, 3 or 4 covalently-linked
monomers, each monomer having a structure independently of formula
I: ##STR00228## wherein each C1-C24 is independently, optionally
substituted: with a hydrocarbon or heterohydrocarbon or
heteroatom-containing functional group, or with an
optionally-substituted alkyl or heteroalkyl, alkenyl or
heteroalkenyl, alkynyl or heteroalkynyl, alkoxyl or heteroalkoxy,
including cyclic and substituted forms of each, aryl and
heteroaryl, wherein hetero-forms comprise 1-4 heteroatoms such as
N, O, P and S, or with a functional group selected from hydroxyl,
haloformyl, carbonyl, aldehyde, carboxyl, ester, acetal,
carboxamide, hydroperoxyl, epoxide, peroxide, oxime, amine, imine,
imide, amide, quaternary ammonium salt, amine oxide, azide, azo,
diazo, azido, aziridine, diaziridine, hydrazine, hydrzone,
aldimine, isocyanide, isocyanate, isothiocyanate, cyanate, nitrate,
nitrile, nitrite, nitride, nitro, nitroso, silane, alkylsilane,
siloxane, halosilane, phosphine, phosphorite, phosphate,
thiophosphonate, quaternary phosphonium salt, phosphono,
phosphides, sulfide, sulfite, sulfonate, thiocyanate, thiosulfate,
sulfoxide, sulfimide, sulfone, sulfoximines, sulfonium, and
sulfhydryl, or with substituted or unsubstituted (C1-C4)alkyl,
(C2-C4)alkenyl, (C6-C8)alkenyl, (C2-C4)alkynyl, (C6-C8)alkynyl,
(C1-C4)alkoxyl, (C6-C8)alkoxyl, 3-oxetanyloxy,
3-tetrahydrofuranyloxy, Cl, F, fluoro-substituted (C1-C2)alkyl,
(C1-C4)alkyl-SO.sub.2--, (C3-C6)cycloalkyl, or a (C5-C6)heterocycle
having 1 or 2 heteroatoms each independently selected from N, O, P,
Si or S.
23. The polymeric bile acid of claim 22, wherein each substituent
is independently: OH, OAc (Ac.dbd.COCH.sub.3, Acetate), OBz
(Bz=COPh, benzoyl), OBn (Bn=benzyl), OTs (Ts=tosyl,
p-toluenesulfonyl), OMs (Ms=methanesulfonyl), OSiR.sub.3, OTf
(Tf=trifluoromethanesulfonyl), OTHP (O-tetrahydropyran), OCOR, OR,
NH.sub.2, NHBoc (Boc=tert-butyloxycarbonyl), NHAc
(Ac.dbd.COCH.sub.3, Acetate), NHBz (Bz=COPh, benzoyl), NHTs
(Ts=tosyl, p-toluenesulfonyl), NHTf (Tf=Trifluoromethanesulfonyl),
NHMs (Ms=methanesulfonyl), NHSiR.sub.3, NHR; NBn.sub.2 (Bn=benzyl),
NTf.sub.2 (Tf=trifluoromethanesulfonyl); NHCOR, NR1R2; --N.sub.2,
--N.sub.3; phosphoric acid (--OP(O)(OH).sub.2, sulfuric
acids(--OSO.sub.2OH) or carboxylic acid (--OCOH), carboxylic ester
(--OCOR); phosphorite(--OP(O).sub.mO--) or phosphate
(--OP(OR).sub.mO--), sulfonate, sulfate(--OS(O).sub.mO--), sulfite
(--OS(OR).sub.mO--), disulfite or pyrosulfate, in which m is equal
to 0, 1 and 2; wherein each R, R1 and R2 is independently H, an
alkyl, aryl, alkenyl, alkynyl, carbonyl, or ether radical having up
to 15 carbon atoms, which is branched or unbranched, a cyclo-alkyl,
aryl, alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15
carbon atoms or a phenyl or benzyl radical, which are unsubstituted
or mono-, bis- or trisubstituted by halide, (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine), or an alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether radical having up to 15 carbon
atoms, which is branched or unbranched, a cyclo-(alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15 carbon
atoms, a phenyl or benzyl radical, which are unsubstituted or
mono-, bis-, tri-substituted by halide, (C1-C4)-alkyl,
(C1-C4)-alkoxyl or (C1-C6)-alkylamine), or heteroatom.
24. The polymeric bile acid of claim 22, wherein each of C3, C6, C7
and C24 is independently, optionally substituted with: OH, OAc
(Ac.dbd.COCH.sub.3, Acetate), OBz (Bz=COPh, benzoyl), OBn
(Bn=benzyl), OTs (Ts=tosyl, p-toluenesulfonyl), OMs
(Ms=methanesulfonyl), OSiR.sub.3, OTf
(Tf=trifluoromethanesulfonyl), OTHP (O-tetrahydropyran), OCOR, OR,
NH.sub.2, NHBoc (Boc=tert-butyloxycarbonyl), NHAc
(Ac.dbd.COCH.sub.3, Acetate), NHBz (Bz=COPh, benzoyl), NHTs
(Ts=tosyl, p-toluenesulfonyl), NHTf (Tf=Trifluoromethanesulfonyl),
NHMs (Ms=methanesulfonyl), NHSiR.sub.3, NHR; NBn.sub.2 (Bn=benzyl),
NTf.sub.2 (Tf=trifluoromethanesulfonyl); NHCOR, NR1R2; --N.sub.2,
--N.sub.3; phosphoric acid (--OP(O)(OH).sub.2, sulfuric
acids(--OSO.sub.2OH) or carboxylic acid (--OCOH), carboxylic ester
(--OCOR); phosphorite(--OP(O).sub.mO--) or phosphate
(--OP(OR).sub.mO--), sulfonate, sulfate(--OS(O).sub.mO--), sulfite
(--OS(OR).sub.mO--), disulfite or pyrosulfate, in which m is equal
to 0, 1 and 2; wherein each R, R1 and R2 is independently H, an
alkyl, aryl, alkenyl, alkynyl, carbonyl, or ether radical having up
to 15 carbon atoms, which is branched or unbranched, a cyclo-alkyl,
aryl, alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15
carbon atoms or a phenyl or benzyl radical, which are unsubstituted
or mono-, bis- or trisubstituted by halide, (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine), or an alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether radical having up to 15 carbon
atoms, which is branched or unbranched, a cyclo-(alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15 carbon
atoms, a phenyl or benzyl radical, which are unsubstituted or
mono-, bis-, tri-substituted by halide, (C1-C4)-alkyl,
(C1-C4)-alkoxyl or (C1-C6)-alkylamine), or heteroatom
25. The polymeric bile acid of claim 22, wherein C24 is substituted
with: --CH.sub.2OH, --COOM, in which M is an alkali metal, alkaline
earth metal or quaternary ammonium ion; or
--CONHCH.sub.2CH.sub.2SO.sub.3H, --CONHRSO.sub.3H or --COOH,
--COOR, --CONH.sub.2, --CH.sub.2NH.sub.2, --CONH or NRR, wherein
each R is independently H, an alkyl, aryl, alkenyl or alkynyl
radical having up to 15 carbon atoms, which is branched or
unbranched, a cycloalkyl radical having 3 to 15 carbon atoms or a
phenyl or benzyl radical, which are unsubstituted or mono-, bis- or
trisubstituted by halide (e.g. F, Cl, Br), (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine).
26. The polymeric bile acid of claim 22, wherein each of C3, C6 and
C7 is independently, optionally substituted with: TABLE-US-00024
--OH, --OAc (Ac = COCH.sub.3, Acetate), --NH.sub.2, --NHBoc (Boc =
tert- --OBz (Bz = COPh, benzoyl), BUtyloxycarbonyl), --OBn (Bn =
benzyl), --NHAc (Ac = COCH.sub.3, Acetate), --OTs (Ts = Tosyl,
p-toluenesulfonyl), --NHBz (Bz = COPh, benzoyl), --OMs (Ms =
methanesulfonyl), - --NHTs (Ts = Tosyl, p-toluenesulfonyl), --OR,
--OSiR.sub.3, --NHTf (Tf = Trifluoromethanesulfonyl), --OTr(Tr =
Triphenylmethyl), --NHMs (Ms = methanesulfonyl), --OTf (Tf =
Trifluoromethanesulfonyl), --NBn.sub.2 (Bn = Benzyl) or --OTHP (THP
= tetrahydropyran), --NR1R2, -carbonyl --N.sub.3, -alkyl
wherein each R, R1 and R2 is independently alkyl, aryl, alkenyl or
alkynyl radical having up to 15 carbon atoms, which is branched or
unbranched, a cycloalkyl radical having 3 to 15 carbon atoms or a
phenyl or benzyl radical, which are unsubstituted or mono-, bis- or
trisubstituted by halide (e.g. F, Cl, Br), (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine).
27. A method of treating HBV or HDV infection or inhibiting human
sodium taurocholate co-transporting polypeptide (hNTCP) comprising
administering to a person in need thereof a polymeric bile acid or
salt thereof, wherein the polymeric bile acid comprises 2, 3 or 4
covalently-linked monomers, each monomer having a structure
independently of formula I: ##STR00229## wherein each C1-C24 is
independently, optionally substituted: with a hydrocarbon or
heterohydrocarbon or heteroatom-containing functional group, or
with an optionally-substituted alkyl or heteroalkyl, alkenyl or
heteroalkenyl, alkynyl or heteroalkynyl, alkoxyl or heteroalkoxy,
including cyclic and substituted forms of each, aryl and
heteroaryl, wherein hetero-forms comprise 1-4 heteroatoms such as
N, O, P and S, or with a functional group selected from hydroxyl,
haloformyl, carbonyl, aldehyde, carboxyl, ester, acetal,
carboxamide, hydroperoxyl, epoxide, peroxide, oxime, amine, imine,
imide, amide, quaternary ammonium salt, amine oxide, azide, azo,
diazo, azido, aziridine, diaziridine, hydrazine, hydrzone,
aldimine, isocyanide, isocyanate, isothiocyanate, cyanate, nitrate,
nitrile, nitrite, nitride, nitro, nitroso, silane, alkylsilane,
siloxane, halosilane, phosphine, phosphorite, phosphate,
thiophosphonate, quaternary phosphonium salt, phosphono,
phosphides, sulfide, sulfite, sulfonate, thiocyanate, thiosulfate,
sulfoxide, sulfimide, sulfone, sulfoximines, sulfonium, and
sulfhydryl, or with substituted or unsubstituted (C1-C4)alkyl,
(C2-C4)alkenyl, (C6-C8)alkenyl, (C2-C4)alkynyl, (C6-C8)alkynyl,
(C1-C4)alkoxyl, (C6-C8)alkoxyl, 3-oxetanyloxy,
3-tetrahydrofuranyloxy, Cl, F, fluoro-substituted (C1-C2)alkyl,
(C1-C4)alkyl-SO.sub.2--, (C3-C6)cycloalkyl, or a (C5-C6)heterocycle
having 1 or 2 heteroatoms each independently selected from N, O, P,
Si or S.
28. The method of claim 27, wherein each substituent is
independently: OH, OAc (Ac.dbd.COCH.sub.3, Acetate), OBz (Bz=COPh,
benzoyl), OBn (Bn=benzyl), OTs (Ts=tosyl, p-toluenesulfonyl), OMs
(Ms=methanesulfonyl), OSiR.sub.3, OTf
(Tf=trifluoromethanesulfonyl), OTHP (O-tetrahydropyran), OCOR, OR,
NH.sub.2, NHBoc (Boc=tert-butyloxycarbonyl), NHAc
(Ac.dbd.COCH.sub.3, Acetate), NHBz (Bz=COPh, benzoyl), NHTs
(Ts=tosyl, p-toluenesulfonyl), NHTf (Tf=Trifluoromethanesulfonyl),
NHMs (Ms=methanesulfonyl), NHSiR.sub.3, NHR; NBn.sub.2 (Bn=benzyl),
NTf.sub.2 (Tf=trifluoromethanesulfonyl); NHCOR, NR1R2; --N.sub.2,
--N.sub.3; phosphoric acid (--OP(O)(OH).sub.2, sulfuric
acids(--OSO.sub.2OH) or carboxylic acid (--OCOH), carboxylic ester
(--OCOR); phosphorite(--OP(O).sub.mO--) or phosphate
(--OP(OR).sub.mO--), sulfonate, sulfate(--OS(O).sub.mO--), sulfite
(--OS(OR).sub.mO--), disulfite or pyrosulfate, in which m is equal
to 0, 1 and 2; wherein each R, R1 and R2 is independently H, an
alkyl, aryl, alkenyl, alkynyl, carbonyl, or ether radical having up
to 15 carbon atoms, which is branched or unbranched, a cyclo-alkyl,
aryl, alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15
carbon atoms or a phenyl or benzyl radical, which are unsubstituted
or mono-, bis- or trisubstituted by halide, (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine), or an alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether radical having up to 15 carbon
atoms, which is branched or unbranched, a cyclo-(alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15 carbon
atoms, a phenyl or benzyl radical, which are unsubstituted or
mono-, bis-, tri-substituted by halide, (C1-C4)-alkyl,
(C1-C4)-alkoxyl or (C1-C6)-alkylamine), or heteroatom.
29. The method of claim 27, wherein each of C3, C6, C7 and C24 is
independently, optionally substituted with: OH, OAc
(Ac.dbd.COCH.sub.3, Acetate), OBz (Bz=COPh, benzoyl), OBn
(Bn=benzyl), OTs (Ts=tosyl, p-toluenesulfonyl), OMs
(Ms=methanesulfonyl), OSiR.sub.3, OTf
(Tf=trifluoromethanesulfonyl), OTHP (O-tetrahydropyran), OCOR, OR,
NH.sub.2, NHBoc (Boc=tert-butyloxycarbonyl), NHAc
(Ac.dbd.COCH.sub.3, Acetate), NHBz (Bz=COPh, benzoyl), NHTs
(Ts=tosyl, p-toluenesulfonyl), NHTf (Tf=Trifluoromethanesulfonyl),
NHMs (Ms=methanesulfonyl), NHSiR.sub.3, NHR; NBn.sub.2 (Bn=benzyl),
NTf.sub.2 (Tf=trifluoromethanesulfonyl); NHCOR, NR1R2; --N.sub.2,
--N.sub.3; phosphoric acid (--OP(O)(OH).sub.2, sulfuric
acids(--OSO.sub.2OH) or carboxylic acid (--OCOH), carboxylic ester
(--OCOR); phosphorite(--OP(O).sub.mO--) or phosphate
(--OP(OR).sub.mO--), sulfonate, sulfate(--OS(O).sub.mO--), sulfite
(--OS(OR).sub.mO--), disulfite or pyrosulfate, in which m is equal
to 0, 1 and 2; wherein each R, R1 and R2 is independently H, an
alkyl, aryl, alkenyl, alkynyl, carbonyl, or ether radical having up
to 15 carbon atoms, which is branched or unbranched, a cyclo-alkyl,
aryl, alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15
carbon atoms or a phenyl or benzyl radical, which are unsubstituted
or mono-, bis- or trisubstituted by halide, (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine), or an alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether radical having up to 15 carbon
atoms, which is branched or unbranched, a cyclo-(alkyl, aryl,
alkenyl, alkynyl, carbonyl, or ether) radical having 3 to 15 carbon
atoms, a phenyl or benzyl radical, which are unsubstituted or
mono-, bis-, tri-substituted by halide, (C1-C4)-alkyl,
(C1-C4)-alkoxyl or (C1-C6)-alkylamine), or heteroatom
30. The method of claim 27, wherein C24 is substituted with:
--CH.sub.2OH, --COOM, in which M is an alkali metal, alkaline earth
metal or quaternary ammonium ion; or
--CONHCH.sub.2CH.sub.2SO.sub.3H, --CONHRSO.sub.3H or --COOH,
--COOR, --CONH.sub.2, --CH.sub.2NH.sub.2, --CONH or NRR, wherein
each R is independently H, an alkyl, aryl, alkenyl or alkynyl
radical having up to 15 carbon atoms, which is branched or
unbranched, a cycloalkyl radical having 3 to 15 carbon atoms or a
phenyl or benzyl radical, which are unsubstituted or mono-, bis- or
trisubstituted by halide (e.g. F, Cl, Br), (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine).
31. The method of claim 27, wherein each of C3, C6 and C7 is
independently, optionally substituted with: TABLE-US-00025 --OH,
--OAc (Ac = COCH.sub.3, Acetate), --NH.sub.2, --NHBoc (Boc = tert-
--OBz (Bz = COPh, benzoyl), BUtyloxycarbonyl), --OBn (Bn = benzyl),
--NHAc (Ac = COCH.sub.3, Acetate), --OTs (Ts = Tosyl,
p-toluenesulfonyl), --NHBz (Bz = COPh, benzoyl), --OMs (Ms =
methanesulfonyl), - --NHTs (Ts = Tosyl, p-toluenesulfonyl), --OR,
--OSiR.sub.3, --NHTf (Tf = Trifluoromethanesulfonyl), --OTr(Tr =
Triphenylmethyl), --NHMs (Ms = methanesulfonyl), --OTf (Tf =
Trifluoromethanesulfonyl), --NBn.sub.2 (Bn = Benzyl) or --OTHP (THP
= tetrahydropyran), --NR1R2, -carbonyl --N.sub.3, -alkyl
wherein each R, R1 and R2 is independently alkyl, aryl, alkenyl or
alkynyl radical having up to 15 carbon atoms, which is branched or
unbranched, a cycloalkyl radical having 3 to 15 carbon atoms or a
phenyl or benzyl radical, which are unsubstituted or mono-, bis- or
trisubstituted by halide (e.g. F, Cl, Br), (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine).
32. The method of claim 27, wherein the polymeric bile acid is in a
predetermined, unit dosage, effective amount.
33. The method of claim 27, further comprising administering to the
person a second, different HBV or HDV medicament.
34. The method of claim 27, further comprising detecting a
resultant inhibition of hNTCP or HBV or HDV infection.
35. A composition comprising a polymeric bile acid or salt thereof,
coformulated or copackaged or coadministered with a second,
different HBV or HDV medicament, wherein the polymeric bile acid
comprises 2, 3 or 4 covalently-linked monomers, each monomer having
a structure independently of formula I: ##STR00230## wherein each
C1-C24 is independently, optionally substituted: with a hydrocarbon
or heterohydrocarbon or heteroatom-containing functional group, or
with an optionally-substituted alkyl or heteroalkyl, alkenyl or
heteroalkenyl, alkynyl or heteroalkynyl, alkoxyl or heteroalkoxy,
including cyclic and substituted forms of each, aryl and
heteroaryl, wherein hetero-forms comprise 1-4 heteroatoms such as
N, O, P and S, or with a functional group selected from hydroxyl,
haloformyl, carbonyl, aldehyde, carboxyl, ester, acetal,
carboxamide, hydroperoxyl, epoxide, peroxide, oxime, amine, imine,
imide, amide, quaternary ammonium salt, amine oxide, azide, azo,
diazo, azido, aziridine, diaziridine, hydrazine, hydrzone,
aldimine, isocyanide, isocyanate, isothiocyanate, cyanate, nitrate,
nitrile, nitrite, nitride, nitro, nitroso, silane, alkylsilane,
siloxane, halosilane, phosphine, phosphorite, phosphate,
thiophosphonate, quaternary phosphonium salt, phosphono,
phosphides, sulfide, sulfite, sulfonate, thiocyanate, thiosulfate,
sulfoxide, sulfimide, sulfone, sulfoximines, sulfonium, and
sulfhydryl, or with substituted or unsubstituted (C1-C4)alkyl,
(C2-C4)alkenyl, (C6-C8)alkenyl, (C2-C4)alkynyl, (C6-C8)alkynyl,
(C1-C4)alkoxyl, (C6-C8)alkoxyl, 3-oxetanyloxy,
3-tetrahydrofuranyloxy, Cl, F, fluoro-substituted (C1-C2)alkyl,
(C1-C4)alkyl-SO.sub.2--, (C3-C6)cycloalkyl, or a (C5-C6)heterocycle
having 1 or 2 heteroatoms each independently selected from N, O, P,
Si or S.
36. The composition of claim 35, wherein the medicament is
lamivudine (Epivir), adefovir (Hepsera), tenofovir (Viread),
telbivudine (Tyzeka), entecavir (Baraclude), bosentan, oxysterol,
ezetimibe, reserpine, rosuvastatin or bromsulphthalein.
Description
INTRODUCTION
[0001] More than one third of the world population has been
infected by Hepatitis B virus (HBV), 240 million people are
presently chronically infected, and 15 million are also infected
with Hepatitis D virus (HDV). HBV infection and related diseases
result in about one million deaths annually.
[0002] We previously identified sodium taurocholate cotransporting
polypeptide (NTCP), a key bile salt transporter of hepatocytes, as
a functional receptor for both HBV and HDV, and we showed that bile
acids could inhibit HBV and HDV infection (Yan et al. Elife
1:e00049, 2012; Yan et al. J Virol 87:7977-7991, 2013; Yan et al. J
Virol 88:3273-3284, 2014). Other NTCP inhibitors have also since
been reported to interfere with HBV and HDV infection, including
irbesartan, ezetimibe, ritonavir(6) and CSA (e.g Blanchet et al.,
Antiviral research 106:111-115, 2014.
[0003] We synthesized bile acid derivatives and used our human
hepatoma HepG2 cells complemented with human NTCP (HepG2-NTCP) cell
culture system to evaluate their inhibitory effect for viral
infection and substrate transporting. By carefully quantitative
analysis we observed anomalous HBV inhibitory activity in some
batches of bile acids, and investigated the source of these
activity discrepancies. We determined that some batches had
contaminating polymeric bile salt bi-products, and we resolved that
these impurities had higher specific activity than the
corresponding normal bile salt. We then prepared purified batches
of a wide range of polymeric bile salts and confirmed enhanced
activity of the polymeric forms across a variety of monomers and
linkages. Accordingly, we disclose uses and compositions of
polymeric bile acid derivatives (PBADs) to inhibit HBV and HDV
infection by targeting hNTCP.
SUMMARY OF THE INVENTION
[0004] The invention provides methods and compositions for treating
HBV or HDV infection or inhibiting human sodium taurocholate
co-transporting polypeptide (hNTCP). In one aspect the method
comprises administering to a person in need thereof a polymeric
bile acid or salt thereof. The method may comprise the antecedent
step of determining that the person is in need thereof, and/or the
subsequent step of detecting a resultant therapeutic effect.
[0005] In embodiments:
[0006] (i) the polymeric bile acid comprises 2, 3 or 4
covalently-linked monomers, each monomer having a structure
independently of formula I:
##STR00001##
[0007] wherein each C1-C24 is independently optionally substituted
with a hydrocarbon or heterohydrocarbon or heteroatom-containing
functional group (non-hydrogen substituent), or with an
optionally-substituted alkyl or heteroalkyl, alkenyl or
heteroalkenyl, alkynyl or heteroalkynyl, alkoxyl or heteroalkoxyl,
including cyclic and substituted forms of each, aryl and
heteroaryl, wherein hetero-forms comprise 1-4 heteroatoms such as
N, O, P and S, or with substituted or unsubstituted (C1-C4)alkyl,
(C2-C4)alkenyl, (C6-C8)alkenyl, (C2-C4)alkynyl, (C6-C8)alkynyl,
(C1-C4)alkoxyl, (C6-C8)alkoxyl, 3-oxetanyloxy,
3-tetrahydrofuranyloxy, Cl, F, fluoro-substituted (C1-C2)alkyl,
(C1-C4)alkyl-SO.sub.2--, (C3-C6)cycloalkyl, or a (C5-C6)heterocycle
having 1 or 2 heteroatoms each independently selected from N, O, P
or S, or with a functional group such as hydroxyl, haloformyl,
carbonyl, aldehyde, carboxyl, ester, acetal, carboxamide,
hydroperoxyl, epoxide, peroxide, oxime, amine (including primary,
secondary and tertiary amines), amide, imine, imide, quaternary
ammonium salt, amine oxide, azide, azo, aldimine, isocyanide,
isocyanate, isothiocyanate, diazo, azido, aziridine, diaziridine,
hydrazine, hydrzone, cyanate, nitrate, nitrile, nitrite, nitride,
nitro, nitroso, silane, alkylsilane, siloxane, halosilane,
phosphine, phosphorite, phosphate, thiophosphonate, quaternary
phosphonium salt, phosphono, phosphides, sulfide, sulfite,
sulfonate, thiocyanate, thiosulfate, sulfoxide, sulfimide, sulfone,
sulfoximines, sulfonium, and sulfhydryl.
[0008] (ii) each substituent is independently:
OH, OAc (Ac.dbd.COCH.sub.3, Acetate), OBz (Bz=COPh, benzoyl), OBn
(Bn=benzyl), OTs (Ts=tosyl, p-toluenesulfonyl), OMs
(Ms=methanesulfonyl), OSiR.sub.3, OTf
(Tf=trifluoromethanesulfonyl), OTHP (THP, tetrahydropyran), OCOR,
OR, NH.sub.2, NHBoc (Boc=tert-butyloxycarbonyl), NHAc
(Ac.dbd.COCH.sub.3, Acetate), NHBz (Bz=COPh, benzoyl), NHTs
(Ts=tosyl, p-toluenesulfonyl), NHTf (Tf=Trifluoromethanesulfonyl),
NHMs (Ms=methanesulfonyl), NHSiR.sub.3, NHR; NBn.sub.2 (Bn=benzyl),
NTf.sub.2 (Tf=trifluoromethanesulfonyl) NHCOR and NR1R2),
--N.sub.2, --N.sub.3; phosphoric acid (--OP(O)(OH).sub.2, sulfuric
acids(--OSO.sub.2OH) or carboxylic acid (--OCOH), carboxylic ester
(--OCOR); phosphorite(--OP(O).sub.mO--) or phosphate
(--OP(OR).sub.mO--), sulfonate, sulfate(--OS(O).sub.mO--), sulfite
(--OS(OR).sub.mO--), disulfite or pyrosulfate, in which m is equal
to 0, 1 and 2; wherein each R, R1 and R2 is independently H, an
alkyl, aryl, alkenyl, alkynyl, carbonyl, or ether radical having up
to 15 carbon atoms, which is branched or unbranched, a
cyclo-(alkyl, aryl, alkenyl, alkynyl, carbonyl, or ether) radical
having 3 to 15 carbon atoms or a phenyl or benzyl radical, which
are unsubstituted or mono-, bis- or trisubstituted by halide,
(C1-C4)-alkyl or (C1-C4)-alkoxyl) or (C1-C6)-alkylamine), ethylene
oxide,
[0009] or an alkyl, aryl, alkenyl or alkynyl radical having up to
15 carbon atoms, which is branched or unbranched, a cycloalkyl
radical having 3 to 15 carbon atoms, a phenyl or benzyl radical,
which are unsubstituted or mono-, bis-, tri-substituted by halide,
(C1-C4)-alkyl, (C1-C4)-alkoxyl or (C1-C6)-alkylamine), or
heteroatom (e.g. --SO.sub.2OH, F, Cl, Br, S).
[0010] (iii) each of C3, C6, C7 and C24 is independently
substituted with:
[0011] a substituent of claim 2; or
[0012] a substituent of claim 3; or
[0013] --NR1R2, --OR or --COR, wherein each R, R1 and R2 is
independently H or a substituent of claim 2;
[0014] (iv) C24 is substituted with:
[0015] --CH.sub.2OH, --COOM, in which M is an alkali metal,
alkaline earth metal or quaternary ammonium ion; or
[0016] --CONHCH.sub.2CH.sub.2SO.sub.3H, --CONHRSO.sub.3H or
[0017] --COOH, --COOR, --CONH.sub.2, --CH.sub.2NH.sub.2, --CONH or
NRR,
[0018] wherein each R is independently H, an alkyl, aryl, alkenyl
or alkynyl radical having up to 15 carbon atoms, which is branched
or unbranched, a cycloalkyl radical having 3 to 10 carbon atoms or
a phenyl or benzyl radical, which are unsubstituted or mono-, bis-
or trisubstituted by halide (e.g. F, Cl, Br), (C1-C4)-alkyl or
(C1-C4)-alkoxyl) or (C1-C6)-alkylamine),
[0019] (v) each of C3, C6 and C7 is independently substituted
with:
TABLE-US-00001 --OH, --OAc (Ac = COCH.sub.3, Acetate), --NH.sub.2,
--NHBoc (Boc = tert-BUtyloxycarbonyl), --OBz (Bz = COPh, benzoyl),
--NHAc (Ac = COCH.sub.3, Acetate), --OBn (Bn = benzyl), --NHBz (Bz
= COPh, benzoyl), --OTs (Ts = Tosyl, p-toluenesulfonyl), --NHTs (Ts
= Tosyl, p-toluenesulfonyl), --OMs (Ms = methanesulfonyl), - --NHTf
(Tf = Trifluoromethanesulfonyl), --OR, --OSiR.sub.3, --NHMs (Ms =
methanesulfonyl), --OTr(Tr = Triphenylmethyl), --NBn.sub.2 (Bn =
Benzyl) or --OTf (Tf = Trifluoromethanesulfonyl), --NR1R2, --OTHP
(THP = tetrahydropyran), --N.sub.3, -carbonyl -alkyl
[0020] wherein each R, R1 and R2 is independently alkyl, aryl,
alkenyl or alkynyl radical having up to 15 carbon atoms, which is
branched or unbranched, a cycloalkyl radical having 3 to 15 carbon
atoms or a phenyl or benzyl radical, which are unsubstituted or
mono-, bis- or trisubstituted by halide (e.g. F, Cl, Br),
(C1-C4)-alkyl or (C1-C4)-alkoxyl) or (C1-C6)-alkylamine);
[0021] (vi) the monomers a linked by 1 or 2 or 3 linkers, each
independently L, wherein L is:
[0022] (a) alkyl, aryl, alkenyl or alkynyl radical having up to 15
carbon atoms, which is branched or unbranched, optionally
substituted and optionally heteroatom containing;
[0023] (b) a cycloalkyl radical having 3 to 15 carbon atoms, or a
phenyl or benzyl radical, which are unsubstituted or mono-, bis-,
tri-substituted by halide (e.g. F, Cl, Br), (C1-C4)-alkyl,
(C1-C4)-alkoxyl or (C1-C6)-alkylamine); or
[0024] (c) a contiguous chain of between 2 and 200 atoms,
preferably between 4 and 100 atoms, more preferably between 4 and
25 atoms, and a MW between 20 and 2K D, preferably between 40 and
1K D, more preferably between 56 and 1K D. L maybe bisymmetrical,
or nonsymmetrical, and may link different, isometric or identical
M1 and M2, typically has spans between about 3 and 3K A, preferably
between about 6 and 2000, more preferably between about 12 and 1000
A, etc.; or
[0025] (d) alkyl, aryl group and heteroatoms, amino acids or other
aminoalkylsulfonic acids bonded with amide or ester bond;
[0026] (vii) the polymeric bile acid comprises a TUDCA moiety;
[0027] (viii) the polymeric bile acid is disclosed in a Table
herein;
[0028] (ix) the polymeric bile acid is in a predetermined, unit
dosage, effective amount.
[0029] (x) the method further comprises administering to the person
a second, different HBV or HDV medicament, and/or detecting a
resultant inhibition of hNTCP or HBV or HDV infection.
[0030] In another aspect the invention provides compositions
comprising a polymeric bile acid or salt thereof, coformulated or
copackaged or coadministered with a second, different HBV or HDV
medicament;
[0031] In embodiments, the polymeric bile acid is a generally- or
specifically-disclosed compound herein, including a polymeric acid
which comprises 2, 3 or 4 covalently-linked monomers, each monomer
having a structure independently of formula I (supra); and/or the
medicament is lamivudine (Epivir), adefovir (Hepsera), tenofovir
(Viread), telbivudine (Tyzeka), entecavir (Baraclude), bosentan,
oxysterol, ezetimibe, reserpine, rosuvastatin, or
bromsulphthalein.
[0032] The invention encompasses all combination of the particular
embodiments recited herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 Schematic illustration of the assays to study drug
candidate potency and mechanism.
[0034] FIG. 2 PBAD inhibited NTCP-mediated HBV and HDV infection.
(A-B) HepG2-NTCP infected with HBV (A) and a control virus
VSV-HBeAg (B) in the presence of indicated PBADs with different
concentrations. On day 5 post infection (dpi), the level of
secreted HBeAg was detected by ELISA. (C) HepG2-NTCP infected with
HDV in the presence of indicated PBADs with different
concentrations. At day 5 post infection, the intracellular delta
antigen were stained with FITC conjugated 4G5 and nuclear stained
with DAPI.
[0035] FIG. 3 PBAD inhibited HBV infection at the entry level.
HepG2-NTCP cells were incubated with indicated PBADs at different
concentrations before (A) or post (B) HBV infection. The treatment
lasted for 24 hrs. The level of secreted HBeAg was detected by
ELISA at 5 dpi.
[0036] FIG. 4 Some PBADs significantly blocked substrate uptake by
NTCP. (A) [3H] taurocholate uptake assay was conducted on
HepG2-NTCP cells in the presence of indicated chemicals or myr47 (a
peptide corresponding to the first 47 amino acids of the L envelope
protein and with a myristoylation modification at the N-terminus).
(B) Dose dependent inhibition of [3H]Taurocholate uptake by
3UT(NQL-012), 3TPT(NQL-018), DiU(NQL-009) and myr47. The indicated
chemicals together with the [3H]taurocholate were added on
HepG2-NTCP cells and incubated for 10 min, the [3H] taurocholate
uptake efficiency was then determined. (C) IC50 of 3UT(NQL-012),
3TPT(NQL-018) and myr47 for inhibiting NTCP-mediated [3H]
taurocholate uptake. HepG2-NTCP cells were pretreated with
indicated concentrations of 3UT(NQL-012), 3TPT(NQL-018) or Myr47
for 2 hrs before uptake assay.
[0037] FIG. 5 IC50 of PBAD for inhibiting HBV infection on
HepG2-NTCP cells. HepG2-NTCP cells were infected by HBV in the
presence of indicated PBADs at different concentrations. The level
of HBeAg in the medium was examined at 5 dpi using ELISA.
[0038] FIG. 6 Toxicity of 3UT(NQL-012), DiU(NQL-009) and
3TPT(NQL-018) in cell cultures. HepG2-NTCP cells were incubated
with 3UT(NQL-012), DiU(NQL-009) or 3TPT(NQL-018) at indicated
concentrations for 48 hrs, cell viability was evaluated and images
were captured at 5 dpi.
[0039] FIG. 7 PBAD inhibited viral infection by targeting NTCP and
blocked the interaction between NTCP and the HBV preS1 domain. (A)
Binding of FITC-preS1 peptide to NTCP was blocked by PBAD.
FITC-preS1 binding assay was conducted in the presence of indicated
concentrations of chemicals. (B) Dose dependence assay of
3TPT(NQL-018) and myr47 for inhibiting the binding between
FITC-preS1 peptide and NTCP. The binding was evaluated as the
HepG2-NTCP cells were pre-incubated (upper panel) or co-incubated
(lower panel) with the chemicals for 3 hrs.
[0040] FIG. 8 The influence of the length of linkage group on the
activity of PBADs. We compared the activity of 3TPT(NQL-018)
(bis-ester bond with 5 carbons in the middle) and 3THT (bis-ester
bond with 7 carbons in the middle). HepG2-NTCP cells were infected
by HBV in the presence of indicated concentrations of PBADs. The
level of secreted HBeAg in the medium was examined at 5 dpi, and
the IC50 value of them are indicated in the figures.
[0041] FIG. 9 PBADs dose dependently inhibited HDV infection.
HepG2-NTCP cells were infected with HDV in the presence of
indicated PBADs with different concentrations. At day 6 post
infection, the intracellular delta antigen was stained with FITC
conjugated monoclonal antibody 4G5. HDV infection ratio was
calculated accordingly.
[0042] FIG. 10 The influence of the linkage groups on the in vitro
stability of PBADs. TUDCA, NQL018, NQL044, and NQL055 were
incubated with HBV inocula of different times before viral
infection, and present in the inocula during infection period. The
level of secreted HBeAg was detected by ELISA at 5 dpi. Only NQL018
showed decreased activity after long time incubation.
[0043] FIG. 11 HepaRG cells were infected with HBV in the presence
of indicated PBADs. The level of secreted HBsAg and HBeAg was
detected by ELISA at 5 dpi.
[0044] FIG. 12 Structure activity relationships analysis of
different PBADs across a variety of monomers and linkages.
HepG2-NTCP cells were infected with HBV in the presence of the
indicated PBADs with different concentration. The level of secreted
HBeAg was detected by ELISA at 5-6 dpi.
DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
[0045] Applications include treatment of chronic HBV and HDV
infection, protection against new HBV infection by vertical
transmission and accidental exposure, prevention of HBV recurrence
after liver transplantation and pathologies indicating NTCP
inhibition, such as protecting hepatocytes from uptake of toxic
bile acids or other NTCP transported agents.
[0046] The descriptions of particular embodiments and examples are
provided by way of illustration and not by way of limitation. Those
skilled in the art will readily recognize a variety of noncritical
parameters that could be changed or modified to yield essentially
similar results.
[0047] Unless contraindicated or noted otherwise, in these
descriptions and throughout this specification, the terms "a" and
"an" mean one or more, the term "or" means and/or and
polynucleotide sequences are understood to encompass opposite
strands as well as alternative backbones described herein.
Furthermore, genuses are recited as shorthand for a recitation of
all members of the genus; for example, the recitation of (C1-C3)
alkyl is shorthand for a recitation of all C1-C3 alkyls: methyl,
ethyl and propyl, including isomers thereof.
[0048] Bile acids are steroid acids comprising four rings in a
sterane core, and a side chain off the C17 carbon, typically of 5
or 8 carbons and terminating in a carboxyl group. Bile acids
encompass natural products, such as found in the bile of mammals,
and synthetic derivatives thereof, such as disclosed herein.
[0049] The term "heteroatom" as used herein generally means any
atom other than carbon or hydrogen. Preferred heteroatoms include
oxygen (O), phosphorus (P), sulfur (S), nitrogen (N), silicon (Si)
and halogens, and preferred heteroatom functional groups are
haloformyl, hydroxyl, aldehyde, amine, quaternary ammonium salt,
amine oxide, azo, diazo, azido, aziridine, diaziridine, hydrazine,
hydrzone, carboxyl, cyanyl, thiocyanyl, carbonyl, halo,
hydroperoxyl, epoxide, peroxide, oxime, imine, imide, amide,
aldimine, isocyanide, iscyanate, isothiocyanate, nitrate, nitrile,
nitrite, nitride, nitro, nitroso, phosphate, thiophosphonate,
phosphono, phosphides, quaternary phosphonium salt, silane,
alkylsilane, siloxane, halosilane, sulfide, sulfite, sulfonate,
thiosulfate, sulfonyl, sulfoxide, sulfimide, sulfone, sulfoximines,
sulfonium, and sulfhydryl.
[0050] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
is fully saturated, having the number of carbon atoms designated
(i.e. C1-C8 means one to eight carbons). Examples of alkyl groups
include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,
isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,
cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,
n-hexyl, n-heptyl, n-octyl and the like.
[0051] The term "alkenyl", by itself or as part of another
substituent, means a straight or branched chain, or cyclic
hydrocarbon radical, or combination thereof, which may be mono- or
polyunsaturated, having the number of carbon atoms designated (i.e.
C2-C8 means two to eight carbons) and one or more double bonds.
Examples of alkenyl groups include vinyl, 2-propenyl, crotyl,
2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl)
and higher homologs and isomers thereof.
[0052] The term "alkynyl", by itself or as part of another
substituent, means a straight or branched chain hydrocarbon
radical, or combination thereof, which may be mono- or
polyunsaturated, having the number of carbon atoms designated (i.e.
C2-C8 means two to eight carbons) and one or more triple bonds.
Examples of alkynyl groups include ethynyl, 1- and 3-propynyl,
3-butynyl and higher homologs and isomers thereof.
[0053] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from alkyl, as
exemplified by --CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--.
Typically, an alkyl (or alkylene) group will have from 1 to 24
carbon atoms, with those groups having 10 or fewer carbon atoms
being preferred in the invention. A "lower alkyl" or "lower
alkylene" is a shorter chain alkyl or alkylene group, generally
having eight or fewer carbon atoms.
[0054] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0055] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and from
one to three heteroatoms selected from the group consisting of O,
N, P, Si and S, wherein the nitrogen, sulfur, and phosphorous atoms
may optionally be oxidized and the nitrogen and phosphorous
heteroatom may optionally be quaternized. The heteroatom(s) O, N, P
and S may be placed at any interior position of the heteroalkyl
group. The heteroatom Si may be placed at any position of the
heteroalkyl group, including the position at which the alkyl group
is attached to the remainder of the molecule. Examples include
--CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3,
--CH.sub.2--S.sub.n--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3,
--CH.sub.2--S--S--CH.sub.2--CH.sub.3, and
--CH.sub.2--O--Si(CH.sub.3).sub.3.
[0056] Similarly, the term "heteroalkylene," by itself or as part
of another substituent means a divalent radical derived from
heteroalkyl, as exemplified by
--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied.
[0057] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Accordingly, a cycloalkyl group has the number of carbon atoms
designated (i.e., C3-C8 means three to eight carbons) and may also
have one or two double bonds. A heterocycloalkyl group consists of
the number of carbon atoms designated and from one to three
heteroatoms selected from the group consisting of O, N, P, Si and
S, and wherein the nitrogen and sulfur atoms may optionally be
oxidized and the nitrogen and phosphine heteroatom may optionally
be quaternized. Additionally, for heterocycloalkyl, a heteroatom
can occupy the position at which the heterocycle is attached to the
remainder of the molecule. Examples of cycloalkyl include
cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include
1-(1,2,5,6-tetrahydropyrid-yl), 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
1-piperazinyl, 2-piperazinyl, and the like.
[0058] The terms "halo" and "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include alkyl substituted with halogen
atoms, which can be the same or different, in a number ranging from
one to (2m'+1), where m' is the total number of carbon atoms in the
alkyl group. For example, the term "halo(C1-C4)alkyl" is mean to
include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,
3-bromopropyl, and the like. Thus, the term "haloalkyl" includes
monohaloalkyl (alkyl substituted with one halogen atom) and
polyhaloalkyl (alkyl substituted with halogen atoms in a number
ranging from two to (2m'+1) halogen atoms, where m' is the total
number of carbon atoms in the alkyl group). The term "perhaloalkyl"
means, unless otherwise stated, alkyl substituted with (2m'+1)
halogen atoms, where m' is the total number of carbon atoms in the
alkyl group. For example the term "perhalo(C1-C4)alkyl" is meant to
include trifluoromethyl, pentachloroethyl,
1,1,1-trifluoro-2-bromo-2-chloroethyl and the like.
[0059] The term "acyl" refers to those groups derived from an
organic acid by removal of the hydroxy portion of the acid.
Accordingly, acyl is meant to include, for example, acetyl,
propionyl, butyryl, decanoyl, pivaloyl, benzoyl and the like.
[0060] The term "aryl" means, unless otherwise stated, a
polyunsaturated, typically aromatic, hydrocarbon substituent which
can be a single ring or multiple rings (up to five rings) which are
fused together or linked covalently. Non-limiting examples of aryl
groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl and
1,2,3,4-tetrahydronaphthalene.
[0061] The term heteroaryl," refers to aryl groups (or rings) that
contain from zero to six heteroatoms selected from N, O, and S,
wherein the nitrogen and sulfur atoms are optionally oxidized and
the nitrogen heteroatom are optionally quaternized. A heteroaryl
group can be attached to the remainder of the molecule through a
heteroatom. Non-limiting examples of heteroaryl groups include
1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,
4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,
2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl,
3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl.
[0062] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0063] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") is meant to include both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0064] Substituents for the alkyl and heteroalkyl radicals (as well
as those groups referred to as alkylene, alkenyl, heteroalkylene,
heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl
and heterocycloalkenyl) can be a variety of groups selected from:
--OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR'--SO.sub.2NR''', --NR''CO.sub.2R', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--SO.sub.2R', --SO.sub.2NR'R'', --NR''SO.sub.2R, --CN and
--NO.sub.2, in a number ranging from zero to three, with those
groups having zero, one or two substituents being particularly
preferred. R', R'' and R''' each independently refer to hydrogen,
unsubstituted (C1-C8)alkyl and heteroalkyl, unsubstituted aryl,
aryl substituted with one to three halogens, unsubstituted alkyl,
alkoxy or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R'
and R'' are attached to the same nitrogen atom, they can be
combined with the nitrogen atom to form a 5-, 6- or 7-membered
ring. For example, --NR'R'' is meant to include 1-pyrrolidinyl and
4-morpholinyl. Typically, an alkyl or heteroalkyl group will have
from zero to three substituents, with those groups having two or
fewer substituents being preferred in the invention. More
preferably, an alkyl or heteroalkyl radical will be unsubstituted
or monosubstituted. Most preferably, an alkyl or heteroalkyl
radical will be unsubstituted. From the above discussion of
substituents, one of skill in the art will understand that the term
"alkyl" is meant to include groups such as trihaloalkyl (e.g.,
--CF.sub.3 and --CH.sub.2CF.sub.3).
[0065] Preferred substituents for the alkyl and heteroalkyl
radicals are selected from: --OR', .dbd.O, --NR'R'', --SR',
halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR''CO.sub.2R',
--NR'--SO.sub.2NR''R''', --S(O)R', --SO2R', --SO.sub.2NR'R'',
--NR''SO.sub.2R, --CN and --NO.sub.2, where R' and R'' are as
defined above. Further preferred substituents are selected from:
--OR', .dbd.O, --NR'R'', halogen, --OC(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR''CO.sub.2R',
--NR'--SO.sub.2NR''R''', --SO.sub.2R', --SO.sub.2NR'R'',
--NR''SO.sub.2R, --CN and --NO.sub.2.
[0066] Similarly, substituents for the aryl and heteroaryl groups
are varied and selected from: halogen, --OR', --OC(O)R', --NR'R'',
--SR', --R', --CN, --NO.sub.2, --CO.sub.2R', --CONR'R'', --C(O)R',
--OC(O)NR'R'', --NR''C(O)R', --NR''CO2R', --NR'--C(O)NR''R''',
--NR'--SO.sub.2NR''R''', --NH--C(NH2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--SO.sub.2R', --SO.sub.2NR'R'', --NR''SO.sub.2R, --N.sub.3,
--CH(Ph).sub.2, perfluoro(C1-C4)alkoxy- and perfluoro(C1-C4)alkyl,
in a number ranging from zero to the total number of open valences
on the aromatic ring system; and where R', R'' and R''' are
independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl,
unsubstituted aryl and heteroaryl, (unsubstituted
aryl)-(C1-C4)alkyl and (unsubstituted aryl)oxy-(C1-C4)alkyl. When
the aryl group is 1,2,3,4-tetrahydronaphthalene, it may be
substituted with a substituted or unsubstituted
(C3-C7)spirocycloalkyl group. The (C3-C7)spirocycloalkyl group may
be substituted in the same manner as defined herein for
"cycloalkyl". Typically, an aryl or heteroaryl group will have from
zero to three substituents, with those groups having two or fewer
substituents being preferred in the invention. In one embodiment of
the invention, an aryl or heteroaryl group will be unsubstituted or
monosubstituted. In another embodiment, an aryl or heteroaryl group
will be unsubstituted.
[0067] Preferred substituents for aryl and heteroaryl groups are
selected from: halogen, --OR', --OC(O)R', --NR'R'', --SR', --R',
--CN, --NO.sub.2, --CO.sub.2R', --CONR'R'', --C(O)R',
--OC(O)NR'R'', --NR''C(O)R', --S(O)R', --SO.sub.2R',
--SO.sub.2NR'R'', --NR''SO.sub.2R, --N.sub.3, --CH(Ph).sub.2,
perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, where R' and R''
are as defined above. Further preferred substituents are selected
from: halogen, --OR', --OC(O)R', --NR'R'', --R', --CN, --NO.sub.2,
--CO.sub.2R', --CONR'R'', --NR''C(O)R', --SO.sub.2R',
--SO.sub.2NR'R'', --NR''SO.sub.2R, perfluoro(C1-C4)alkoxy and
perfluoro(C1-C4)alkyl.
[0068] The substituent --CO.sub.2H, as used herein, includes
bioisosteric replacements therefor; see, e.g., The Practice of
Medicinal Chemistry; Wermuth, C. G., Ed.; Academic Press: New York,
1996; p. 203.
[0069] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CH.sub.2)q-U-, wherein T and U are
independently --NH--, --O--, --CH.sub.2-- or a single bond, and q
is an integer of from 0 to 2. Alternatively, two of the
substituents on adjacent atoms of the aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula
-A-(CH2)r-B-, wherein A and B are independently --CH.sub.2--,
--O--, --NH--, --S--, --S(O)--, --S(O).sub.2--, --S(O).sub.2NR'--
or a single bond, and r is an integer of from 1 to 3. One of the
single bonds of the new ring so formed may optionally be replaced
with a double bond. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
--(CH.sub.2)s-X--(CH.sub.2)t-, where s and t are independently
integers of from 0 to 3, and X is --O--, --S--, --S(O)--,
--S(O).sub.2--, or --S(O).sub.2NR'--. The substituent R' in --NR'--
and --S(O).sub.2NR'-- is selected from hydrogen or unsubstituted
(C1-C6)alkyl.
[0070] Preferred substituents are disclosed herein and exemplified
in the tables, structures, examples, and claims, and may be applied
across different compounds of the invention, i.e. substituents of
any given compound may be combinatorially used with other
compounds.
[0071] In particular embodiments applicable substituents are
independently substituted or unsubstituted heteroatom, substituted
or unsubstituted, optionally heteroatom C1-C6 alkyl, substituted or
unsubstituted, optionally heteroatom C2-C6 alkenyl, substituted or
unsubstituted, optionally heteroatom C2-C6 alkynyl, or substituted
or unsubstituted, optionally heteroatom C6-C14 aryl, wherein each
heteroatom is independently oxygen, phosphorus, sulfur or
nitrogen.
[0072] In more particular embodiments, applicable substituents are
independently aldehyde, aldimine, alkanoyloxy, alkoxy,
alkoxycarbonyl, alkyloxy, alkyl, amine, quaternary ammonium salt,
amine oxide, azo, diazo, azido, aziridine, diaziridine, hydrazine,
hydrzone, halogens, carbamoyl, carbonyl, carboxamido, carboxyl,
cyanyl, thiocyanyl, ester, halo, haloformyl, hydroperoxyl,
hydroxyl, epoxide, peroxide, oxime, imine, imide, amide, aldimine,
isocyanide, iscyanate, isothiocyanate, N-tert-butoxycarbonyl,
nitrate, nitrile, nitrite, nitro, nitroso, phosphate,
thiophosphonate, phosphides, phosphono, quaternary phosphonium
salt, silane, alkylsilane, siloxane, halosilane, sulfide, sulfite,
sulfonate, thiosulfate, sulfonyl, sulfo, sulfoxide, sulfimide,
sulfone, sulfoximines, sulfonium, sulfhydryl, thiol, thiocyanyl,
trifluoromethyl or trifluromethyl ether (OCF.sub.3).
[0073] The term "pharmaceutically acceptable salts" is meant to
include salts of the active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituents found on the compounds described herein. When
compounds of the invention contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable base addition salts include sodium,
potassium, calcium, ammonium, organic amino, or magnesium salt, or
a similar salt. When compounds of the invention contain relatively
basic functionalities, acid addition salts can be obtained by
contacting the neutral form of such compounds with a sufficient
amount of the desired acid, either neat or in a suitable inert
solvent. Examples of pharmaceutically acceptable acid addition
salts include those derived from inorganic acids like hydrochloric,
hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, oxalic, maleic, malonic,
benzoic, succinic, suberic, fumaric, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like. Certain
specific compounds of the invention contain both basic and acidic
functionalities that allow the compounds to be converted into
either base or acid addition salts.
[0074] The neutral forms of the compounds may be regenerated by
contacting the salt with a base or acid and isolating the parent
compound in the conventional manner. The parent form of the
compound differs from the various salt forms in certain physical
properties, such as solubility in polar solvents, but otherwise the
salts are equivalent to the parent form of the compound for the
purposes of the invention.
[0075] In addition to salt forms, the invention provides compounds
which are in a prodrug form. Prodrugs of the compounds described
herein are those compounds that undergo chemical changes under
physiological conditions to provide the compounds of the invention.
Additionally, prodrugs can be converted to the compounds of the
invention by chemical or biochemical methods in an ex vivo
environment. For example, prodrugs can be slowly converted to the
compounds of the invention when placed in a transdermal patch
reservoir with a suitable enzyme or chemical reagent. Prodrugs are
often useful because, in some situations, they may be easier to
administer than the parent drug. They may, for instance, be more
bioavailable by oral administration than the parent drug. The
prodrug may also have improved solubility in pharmacological
compositions over the parent drug. A wide variety of prodrug
derivatives are known in the art, such as those that rely on
hydrolytic cleavage or oxidative activation of the prodrug. An
example, without limitation, of a prodrug would be a compound of
the invention which is administered as an ester (the "prodrug"),
but then is metabolically hydrolyzed to the carboxylic acid, the
active entity. Additional examples include peptidyl derivatives of
a compound of the invention.
[0076] Certain compounds of the invention can exist in unsolvated
forms as well as solvated forms, including hydrated forms. In
general, the solvated forms are equivalent to unsolvated forms and
are intended to be encompassed within the scope of the invention.
Certain compounds of the invention may exist in multiple
crystalline or amorphous forms. In general, all physical forms are
equivalent for the uses contemplated by the invention and are
intended to be within the scope of the invention.
[0077] Some of the subject compounds possess asymmetric carbon
atoms (optical centers) or double bonds; the racemates,
diastereomers, geometric isomers and specifically designated or
depicted chirality is preferred and in many cases critical for
optimal activity; however all such isomers are all intended to be
encompassed within the scope of the invention.
[0078] The compounds of the invention may also contain unnatural
proportions of atomic isotopes at one or more of the atoms that
constitute such compounds. For example, the compounds may be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I), Fluorine(.sup.18F, .sup.17 F) or
carbon-14 (.sup.14C, .sup.13C). All isotopic variations of the
compounds of the invention, whether radioactive or not, are
intended to be encompassed within the scope of the invention.
[0079] The term "therapeutically effective amount" refers to the
amount of the subject compound that will elicit, to some
significant extent, the biological or medical response of a tissue,
system, animal or human that is being sought by the researcher,
veterinarian, medical doctor or other clinician, such as when
administered, is sufficient to prevent development of, or alleviate
to some extent, one or more of the symptoms of the condition or
disorder being treated. The therapeutically effective amount will
vary depending on the compound, the disease and its severity and
the age, weight, etc., of the mammal to be treated.
[0080] The invention also provides pharmaceutical compositions
comprising the subject compounds and a pharmaceutically acceptable
excipient, particularly such compositions comprising a unit dosage
of the subject compounds, particularly such compositions copackaged
with instructions describing use of the composition to treat an
applicable disease or condition (herein).
[0081] The compositions for administration can take the form of
bulk liquid solutions or suspensions, or bulk powders. More
commonly, however, the compositions are presented in unit dosage
forms to facilitate accurate dosing. The term "unit dosage forms"
refers to physically discrete units suitable as unitary dosages for
human subjects and other mammals, each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect, in association with a suitable
pharmaceutical excipient. Typical unit dosage forms include
prefilled, premeasured ampules or syringes of the liquid
compositions or pills, tablets, capsules, losenges or the like in
the case of solid compositions. In such compositions, the compound
is usually a minor component (from about 0.1 to about 50% by weight
or preferably from about 1 to about 40% by weight) with the
remainder being various vehicles or carriers and processing aids
helpful for forming the desired dosing form.
[0082] Suitable excipients or carriers and methods for preparing
administrable compositions are known or apparent to those skilled
in the art and are described in more detail in such publications as
Remington's Pharmaceutical Science, Mack Publishing Co, N.J.
(1991). In addition, the compounds may be advantageously used in
conjunction with other therapeutic agents as described herein or
otherwise known in the art, particularly other anti-diabetes or
anti-obesity agents. Hence the compositions may be administered
separately, jointly, or combined in a single dosage unit.
[0083] The amount administered depends on the compound formulation,
route of administration, etc. and is generally empirically
determined in routine trials, and variations will necessarily occur
depending on the target, the host, and the route of administration,
etc. Generally, the quantity of active compound in a unit dose of
preparation may be varied or adjusted from about 1, 3, 10 or 30 to
about 30, 100, 300 or 1000 mg, according to the particular
application. In a particular embodiment, unit dosage forms are
packaged in a multipack adapted for sequential use, such as
blisterpack, comprising sheets of at least 6, 9 or 12 unit dosage
forms. The actual dosage employed may be varied depending upon the
requirements of the patient and the severity of the condition being
treated. Determination of the proper dosage for a particular
situation is within the skill of the art. Generally, treatment is
initiated with smaller dosages which are less than the optimum dose
of the compound. Thereafter, the dosage is increased by small
amounts until the optimum effect under the circumstances is
reached. For convenience, the total daily dosage may be divided and
administered in portions during the day if desired.
[0084] The compounds can be administered by a variety of methods
including, but not limited to, parenteral, topical, oral, or local
administration, such as by aerosol or transdermally, for
prophylactic and/or therapeutic treatment. Also, in accordance with
the knowledge of the skilled clinician, the therapeutic protocols
(e.g., dosage amounts and times of administration) can be varied in
view of the observed effects of the administered therapeutic agents
on the patient, and in view of the observed responses of the
disease to the administered therapeutic agents.
[0085] The therapeutics of the invention can be administered in a
therapeutically effective dosage and amount, in the process of a
therapeutically effective protocol for treatment of the patient.
For more potent compounds, microgram (ug) amounts per kilogram of
patient may be sufficient, for example, in the range of about 1, 10
or 100 ug/kg to about 0.01, 0.1, 1, 10, or 100 mg/kg of patient
weight though optimal dosages are compound specific, and generally
empirically determined for each compound.
[0086] In general, routine experimentation in clinical trials will
determine specific ranges for optimal therapeutic effect, for each
therapeutic, each administrative protocol, and administration to
specific patients will also be adjusted to within effective and
safe ranges depending on the patient condition and responsiveness
to initial administrations. However, the ultimate administration
protocol will be regulated according to the judgment of the
attending clinician considering such factors as age, condition and
size of the patient as well as compounds potency, severity of the
disease being treated. For example, a dosage regimen of the
compounds can be oral administration of from 10 mg to 2000 mg/day,
preferably 10 to 1000 mg/day, more preferably 50 to 600 mg/day, in
two to four (preferably two) divided doses. Intermittent therapy
(e.g., one week out of three weeks or three out of four weeks) may
also be used.
[0087] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein,
including citations therein, are hereby incorporated by reference
in their entirety for all purposes.
EXAMPLES
[0088] PBADs, including 3UT(NQL-012), 7UT(NQL-015), DIU(NQL-009)
and 3TPT(NQL-018), showed greatly improved potency as the inhibitor
of HBV and HDV virus infection. Our data indicate they can inhibit
infection of HBV and HDV on HepG2-NTCP cells with an IC50 down to
less than 50 nM, which is 100 times lower than their common
precursor tauroursodeoxycholic acid (TUDCA). PBADs can also
significantly block the substrate uptake of NTCP so they can also
serve as NTCP transporting inhibitors.
TABLE-US-00002 TABLE A TUDCA/UDCA monomer: ##STR00002##
Ursodeoxycholic acid (UDCA, U) ##STR00003## Tauroursodeoxycholic
acid (TUDCA, T) ##STR00004## Formula (III) 3-T-R ##STR00005##
Formula (IV) 7-T-R ##STR00006## Formula (II) 24-U-R TUDCA/UDCA
Dimer: ##STR00007## ##STR00008## 24-UT amine 24-Ursodeoxycholyl
Tauroursodeoxycholamine ##STR00009## m = 1-7 24-UEU
24-Di-Ursodeoxycholyl-Ester-Ursodeoxycholic acid ##STR00010## X =
NH 24-DIU amine X = O, 24-DIU Ether 24-Ursodeoxycholyl
Ursodeoxycholamine 24-Ursodeoxycholyl Ursodeoxycholyl ether
##STR00011## 24-UAU 24-Ursodeoxycholyl-Amide-Ursodeoxycholic acid
##STR00012## m = 0-7 n = 1-7 X = NH, 24-DIU polyamine X = O, 24-DIU
polyether Poly-24-Ursodeoxycholyl Ursodeoxycholyl amine
Poly-24-Ursodeoxycholyl Ursodeoxycholyl ether ##STR00013## m = 0-7
n = 1-7 X = O, 24-UUPE X = NR, 24-UUPA X = NR, 24-Ursodeoxycholyl
Ursodeoxycholyl PolyAmide X = O, 24-Ursodeoxycholyl Ursodeoxycholyl
PolyEsterr ##STR00014## m = 0-7 n = 1-7 X = NR, 24-PHR UU Amide-1 X
= O, 24-PHR UU Ester-1 Cy = Saturated or Unsaturated ring system
including, any sized Heterocycle or Carbocycles with Cic/Trans;
para-, ortho- or meta-substitution ##STR00015## m = 0-7 n = 1-7 X =
NR, 24-PHR UU Amide-2 X = O, 24-PHR UU Ester-2 Cy = Saturated or
Unsaturated ring system including, any sized Heterocycle or
Carbocycles with Cic/Trans; para-, ortho- or meta-substitution
##STR00016## m = 0-7 n = 1-7 X = NR, 24-PPR UU Amine-3 X = O,
24-PPR UU Ether-3 Cy = Saturated or Unsaturated ring system
including, any sized Heterocycle or Carbocycles with Cic/Trans;
para-, ortho- or meta-substitution ##STR00017## X = NR, 24-PPR UU
Amine-4 X = O, 24-PPR UU Ether-4 Cy = Saturated or Unsaturated ring
system including, any sized Heterocycle or Carbocycles with
Cic/Trans; para-, ortho- or meta-substitution ##STR00018##
##STR00019## 3-TAT 3-Tauroursodeoxycholic
Amide-Tauroursodeoxycholic Acid ##STR00020## X = NR, 3-UT amine X =
O, 3-UT Ether 3-Ursodeoxycholyl Tauroursodeoxycholyl amine
3-Ursodeoxycholyl Tauroursodeoxycholyl ether ##STR00021## m = 1-7
3-UTE 3-Ursodeoxycholyl Tauroursodeoxycholyl Ester ##STR00022## m =
1-7 3-UTA 3-Ursodeoxycholyl Tauroursodeoxycholyl Amide ##STR00023##
m = 0-7 n = 0-7 X = O, 3-UTPE X = NR, 3-UTPA X = NR,
3-Ursodeoxycholyl Tauroursodeoxycholyl PolyAmide X = O,
3-Ursodeoxycholyl Tauroursodeoxycholyl PolyEsterr ##STR00024## X =
O, 3-UTP Ether X = NR, 3-UTP Amine X = NR, 3-Ursodeoxycholyl
Tauroursodeoxycholyl PolyAmide X = O, 3-Ursodeoxycholyl
Tauroursodeoxycholyl PolyEsterr ##STR00025## m = 0-7 n = 1-7 X =
NR, 3-PPR UT Amide X = O, 3-PPR UT Ester Cy = Saturated or
Unsaturated ring system including, any sized Heterocycle or
Carbocycles with Cic/Trans; para-, ortho- or meta-substitution
##STR00026## m = 0-7 n = 1-7 X = NR, 3-PPR UT Amine X = O, 3-PPR UT
Ether Cy = Saturated or Unsaturated ring system including, any
sized Heterocycle or Carbocycles with Cic/Trans; para-, ortho- or
meta-substitution ##STR00027## ##STR00028## X = NR, 7-UT amine X =
O, 7-UT Ether 7-Ursodeoxycholyl Tauroursodeoxycholyl amine
7-Ursodeoxycholyl Tauroursodeoxycholyl ether ##STR00029## 7-TAT
7-Tauroursodeoxycholic Amide-Tauroursodeoxycholic Acid ##STR00030##
m = 1-7 7-UTE 7-Ursodeoxycholyl Tauroursodeoxycholyl Ester
##STR00031## m = 1-7 7-UTA 7-Ursodeoxycholyl Tauroursodeoxycholyl
Amide ##STR00032## X = O, 7-UTPE X = NR, 7-UTPA X = NR,
7-Ursodeoxycholyl Tauroursodeoxycholyl PolyAmide X = O,
7-Ursodeoxycholyl Tauroursodeoxycholyl PolyEster ##STR00033## X =
O, 7-UTP Ether X = NR, 7-UTP Amine X = NR, 7-Ursodeoxycholyl
Tauroursodeoxycholyl PolyAmine X = O, 7-Ursodeoxycholyl
Tauroursodeoxycholyl PolyEther ##STR00034## m = 0-7 n = 1-7 X = NR,
7-PHR UT Amide X = O, 7-PHR UT Ester Cy = Saturated or Unsaturated
ring system including, any sized Heterocycle or Carbocycles with
Cic/Trans; para-, ortho- or meta-substitution ##STR00035## m = 0-7
n = 1-7 X = NR, 7-PPR UT Amine X = O, 7-PPR UT Ether Cy = Saturated
or Unsaturated ring system including, any sized Heterocycle or
Carbocycles with Cic/Trans; para-, ortho- or meta-substitution
##STR00036## ##STR00037## m = 1-7 X = NR, 3-RTT Amide X = O, 3-RTT
Ester ##STR00038## m = 0-5 n = 0-5 X = O, 3-RTTPE X = NR, 3-RTTPA
##STR00039## m = 0-7 n = 1-7 X = NR, 3-Polyalkyne
Tauroursodeoxycholyl Tauroursodeoxycholyl Amine
X = O, 3-Polyalkyne Tauroursodeoxycholyl Tauroursodeoxycholyl Ester
##STR00040## m = 0-7 3-Triazole TT Ether 3-Triazole
Tauroursodeoxycholyl Tauroursodeoxycholyl Ether ##STR00041## m =
0-7 n = 1-7 X = NR, 3-PHR TT Amine X = O, 3-PHR TT Ether Cy =
Saturated or Unsaturated ring system including, any sized
Heterocycle or Carbocycles with Cic/Trans; para-, ortho- or
meta-substitution ##STR00042## m = 0-7 n = 1-7 X = NR, 3-PHR TT
Amide X = O, 3-PHR TT Ester Cy = Saturated or Unsaturated ring
system including, any sized Heterocycle or Carbocycles with
Cic/Trans; para-, ortho- or meta-substitution ##STR00043## 3-Diol
TT 3-Diol Tauroursodeoxycholyl Tauroursodeoxycholyl ##STR00044##
3-Alkene(E/Z) TT 3-Alkene(E/Z) Tauroursodeoxycholyl
Tauroursodeoxycholyl ##STR00045## 3-TT 3-Tauroursodeoxycholyl
Tauroursodeoxycholyl ##STR00046## m = 0-7 n = 1-7 X = NR, 3-PHepR
TT Amine X = O, 3-PHepR TT Ether Cy = Saturated or Unsaturated ring
system including, any sized Heterocycle or Carbocycles with
Cic/Trans; para-, ortho- or meta-substitution ##STR00047## m = 0-7
n = 1-7 X = NR, 3-PHepR TT Amide X = O, 3-PHepR TT Ester Cy =
Saturated or Unsaturated ring system including, any sized
Heterocycle or Carbocycles with Cic/Trans; para-, ortho- or
meta-substitution ##STR00048## m = 0-7 n = 1-7 X = NR,
3-Polyalkene-Tauroursodeoxycholyl Tauroursodeoxycholyl Amine X = O,
3-Polyalkene-Tauroursodeoxycholyl Tauroursodeoxycholyl Ether
##STR00049## m = 0-7 n = 1-7 X = NR, 3-PTne TT Amide X = O, 3-PYne
TT Ester X = NR, 3-Polyalkene-Tauroursodeoxycholyl
Tauroursodeoxycholyl Amide X = O, 3-Polyalkene-Tauroursodeoxycholyl
Tauroursodeoxycholyl Ester ##STR00050## 2,3-pyrazine TT
2,3-pyrazine Tauroursodeoxycholyl Tauroursodeoxycholyl ##STR00051##
m = 0-7 n = 1-7 X = NR, 3-PAlkyne TT Amide X = O, 3-PAlkyne TT
Ester X = NR, 3-Polyalkyne-Tauroursodeoxycholyl
Tauroursodeoxycholyl Amide X = O, 3-Polyalkyne-Tauroursodeoxycholyl
Tauroursodeoxycholyl Ester ##STR00052## m = 1-7 n = 1-3 X = NR,
3-(7-PPhosphate)TT Amide X = O, 3-(7-PPhosphate)TT Ester
3-(7-PolyPhosphate)tauroursodeoxycholyl Tauroursodeoxycholyl Amide
3-(7-PolyPhosphate)tauroursodeoxycholyl Tauroursodeoxycholyl Ester
##STR00053## m = 1-7 n = 1-3 X = NR, 3-(7-PDiphosphate)TT Amide X =
O, 3-(7-PDiphosphate)TT Ester
3-(7-PolyDiphosphate)tauroursodeoxycholyl Tauroursodeoxycholyl
Amide 3-(7-PolyDiphosphate)tauroursodeoxycholyl
Tauroursodeoxycholyl Ester ##STR00054## m = 1-7 X = NR, 3-(7-Diz)TT
Amide X = O, 3-(7-Diz)TT Ester 3-(7-Diz)tauroursodeoxycholyl
Tauroursodeoxycholyl Amide 3-(7-Diz)tauroursodeoxycholyl
Tauroursodeoxycholyl Ester ##STR00055## m = 1-7 X = NR,
3-(7-Didiz)TT Amide X = O, 3-(7-Didiz)TT Ester
3-(7-Didiz)tauroursodeoxycholyl Tauroursodeoxycholyl Amide
3-(7-Didiz)tauroursodeoxycholyl Tauroursodeoxycholyl Ester
##STR00056## m = 1-7 X = NR, 3-(7-Bio)TT Amine X = O, 3-(7-Bio)TT
Ester 3-(7-Bio-7'-Diz)tauroursodeoxycholyl Tauroursodeoxycholyl
Amide 3-(7-Bio-7'-Diz)tauroursodeoxycholyl Tauroursodeoxycholyl
Ester ##STR00057## m = 1-7 X = NR, 3-(7-Bio-7'-Diz)TT Amine X = O,
3-(7-Bio-7'-Diz)TT Ester 3-(7-Bio-7'-Diz)tauroursodeoxycholyl
Tauroursodeoxycholyl Amide 3-(7-Bio-7'-Diz)tauroursodeoxycholyl
Tauroursodeoxycholyl Ester ##STR00058## m = 1-7 3-PRibose TT Ether
3-PolyRibose-Tauroursodeoxycholyl Tauroursodeoxycholyl Ether
##STR00059## m = 1-7 3-PRibose TT Amine
3-PolyRibose-Tauroursodeoxycholyl Tauroursodeoxycholyl Amine
##STR00060## m = 1-7 n = 0-7 o = 1-7 X = NR, 3-Polyol TT Amide X =
O, 3-Polyol TT Ester X = NR, 3-Polyol-Tauroursodeoxycholyl
Tauroursodeoxycholyl Amide X = O, 3-Polyol-Tauroursodeoxycholyl
Tauroursodeoxycholyl Ester ##STR00061## m = 1-7 n = 0-7 o = 1-7 X =
NR, 3-Polyone TT Amide X = O, 3-Polyone TT Ester X = NR,
3-Polyone-Tauroursodeoxycholyl Tauroursodeoxycholyl Amide X = O,
3-Polyone-Tauroursodeoxycholyl Tauroursodeoxycholyl Ester
##STR00062## m = 1-7 X = NR, 3-T Rosuvastatin Amide X = O, 3-T
Rosuvastatin Ester X = NR, 3-Tauroursodeoxycholyl Rosuvastatin
Amide X = O, 3-Tauroursodeoxycholyl Rosuvastatin Ester ##STR00063##
m = 1-7 X = NR, 3-T Reserpine Amide X = O, 3-T Reserpine Ester X =
NR, 3-Tauroursodeoxycholyl Reserpine Amide X = O,
3-Tauroursodeoxycholyl Reserpine Ester ##STR00064## m = 1-5 X = NR,
3-TToestrone Amine X = O, 3-TToestrone Ether X = NR,
3-TauroUrsodeoxycholyl Toestrone Amine X = O,
3-TauroUrsodeoxycholyl Toestrone Ether ##STR00065## m = 1-7 X = NR,
3-T Bromsulphthalein Amide X = O, 3-T Bromsulphthalein Ester X =
NR, 3-Tauroursodeoxycholyl Bromsulphthalein Amide X = O,
3-Tauroursodeoxycholyl Bromsulphthalein Ester ##STR00066## m = 1-7
X = NR, 3-T Adamantanol Amide X = O, 3-T Adamantanol Ester X = NR,
3-Tauroursodeoxycholyl Adamantanol Amide X = O,
3-Tauroursodeoxycholyl Adamantanol Ester ##STR00067## m = 1-5 X =
NR, 3-T Naphthalene Amine X = O, 3-T Naphthalene Ether X = NR,
3-TauroUrsodeoxycholyl Naphthalene Amine X = O,
3-TauroUrsodeoxycholyl Naphthalene Ether ##STR00068## m = 1-7 X =
NR, 3-T CsA Amide X = O, 3-T Csa Ester X = NR,
3-Tauroursodeoxycholyl CsA Amide X = O, 3-Tauroursodeoxycholyl CsA
Ester ##STR00069## m = 1-7 X = NR, 3-T Bosentan Amide X = O, 3-T
Bosentan Ester X = NR, 3-Tauroursodeoxycholyl Bosentan Amide X = O,
3-Tauroursodeoxycholyl Bosentan Ester ##STR00070## m = 1-7 X = NR,
3-T Oxysterol Amide X = O, 3-T Oxysterol Ester X = NR,
3-Tauroursodeoxycholyl Oxysterol Amide X = O,
3-Tauroursodeoxycholyl Oxysterol Ester ##STR00071## m = 1-7 X = NR,
3-T Ezetimibe Amide
X = O, 3-T Ezetimibe Ester X = NR, 3-Tauroursodeoxycholyl Ezetimibe
Amide X = O, 3-Tauroursodeoxycholyl Ezetimibe Ester ##STR00072## m
= 1-7 n = 0-7 o = 1-7 X = NR, 3-(24-Polycyclic
Lactam)TauroUrsodeoxycholyl Tauroursodeoxycholyl Amide X = O,
3-(24-Polycyclic Lactam)TauroUrsodeoxycholyl Tauroursodeoxycholyl
Ester Cy = Saturated or Unsaturated ring system including, any
sized Heterocycle or Carbocycles with Cic/Trans; para-, ortho- or
meta-substitution ##STR00073## m = 1-7 n = 0-7 o = 1-7 q = 1-7 X =
NR, 3-(24-PPRLactam)TT Amine X = O, 3-(24-PPRLactam)TT Ether Cy =
Saturated or Unsaturated ring system including, any sized
Heterocycle or Carbocycles with Cic/Trans; para-, ortho- or
meta-substitution ##STR00074## n = 0-7 o = 1-7 m = 1-7 X = NR,
3-(24-PLactam)TT Amide X = O, 3-(24-PLactam)TT Ester X = NR,
3-(24-PolyLactam)TauroUrsodeoxycholyl Tauroursodeoxycholyl Amide X
= O, 3-(24-PolyLactam)TauroUrsodeoxycholyl Tauroursodeoxycholyl
Ester ##STR00075## m = 1-7 n = 1-7 X = O, Y = NR,
3-(7,24'-PolyEther)UT Amide X = NR, Y = O, 3-(7,24'-PolyAmine)UT
Ester X = O, Y = O, 3-(7,24'-PolyEther)UT Ester X = NR, Y = NR,
3-(7,24'-PolyAmine)UT Amide X = O, Y = NR,
3-(7,24'-PolyEther)Ursodeoxycholyl Tauroursodeoxycholyl Amide X =
O, Y = NR, 3-(7,24'-PolyAmine)Ursodeoxycholyl Tauroursodeoxycholyl
Ester X = O, Y = NR, 3-(7,24'-PolyEther)Ursodeoxycholyl
Tauroursodeoxycholyl Ester X = O, Y = NR,
3-(7,24'-PolyAmine)Ursodeoxycholyl Tauroursodeoxycholyl Amide
##STR00076## n = 0-7 o = 1-7 m = 1-7 X = NR,
3-(24-PolyLactam)TauroUrsodeoxycholyl Tauroursodeoxycholyl Amine X
= O, 3-(24-PolyLactam)TauroUrsodeoxycholyl Tauroursodeoxycholyl
Ether ##STR00077## m = 1-7 n = 1-7 X = O, Y = NR,
3-(7,24'-PolyEster)Ursodeoxycholyl Tauroursodeoxycholyl Amide X =
O, Y = NR, 3-(7,24'-PolyAmine)Ursodeoxycholyl Tauroursodeoxycholyl
Ester X = O, Y = NR, 3-(7,24'-PolyEster)Ursodeoxycholyl
Tauroursodeoxycholyl Ester X = O, Y = NR,
3-(7,24'-PolyAmine)Ursodeoxycholyl Tauroursodeoxycholyl Amide
##STR00078## m = 1-7 n = 1-7 o = 1-7 q = 1-7 X = NR, Y = NR,
3-(24-Polyamine) Ursodeoxycholyl Ursodeoxycholyl Amide X = O, Y =
NR, 3-(24-Polyamine) Ursodeoxycholyl ursodeoxycholyl Ester X = NR,
Y = O, 3-(24-Polyether) Ursodeoxycholyl Ursodeoxycholyl Amide X =
O, Y = O, 3-(24-Polyether) Ursodeoxycholyl ursodeoxycholyl Ester
##STR00079## m = 1-7 n = 1-7 o = 1-7 q = 1-7 X = NR, Y = NR,
3-(24-PolyLactam) Ursodeoxycholyl Ursodeoxycholyl Amine X = O, Y =
NR, 3-(24-PolyLactam) Ursodeoxycholyl ursodeoxycholyl Ether X = NR,
Y = O, 3-(24-PolyLactone) Ursodeoxycholyl Ursodeoxycholyl Amine X =
O, Y = O, 3-(24-PolyLactone) Ursodeoxycholyl ursodeoxycholyl Ether
7-TPT: ##STR00080## 7-TPT Di-7-Tauroursodeoxycholyl Glutarate
##STR00081## 7-TPAT Di-7-Tauroursodeoxycholyl Glutaramide
##STR00082## m = 1-7 7-TT Ester 7-Tauroursodeoxycholyl
Tauroursodeoxycholyl Ester ##STR00083## X = O, 7-TT Diether X = NR,
7-TT Diamine X = NR, 7-TauroUrsodeoxycholyl Tauroursodeoxycholyl
Diamine X = O, 7-TauroUrsodeoxycholyl Tauroursodeoxycholyl Diether
##STR00084## m = 1-7 7-TT Amide 7-Tauroursodeoxycholyl
Tauroursodeoxycholyl Amide ##STR00085## 7-TPT X = NR, 3-TTPA; X =
O, 3-TTPE X = NR, 7-TauroUrsodeoxycholyl Tauroursodeoxycholyl
PolyAmine X = O, 7-TauroUrsodeoxycholyl Tauroursodeoxycholyl
PolyEther ##STR00086## m = 0-7 n = 0-7 X = NR, 7-PL UU Amide X = O,
7-PL UU Ester X = O, 7,24'-PolyLactone Ursodeoxycholyl
Ursodeoxycholyl Amide X = NR, 7,24'-PolyLactam Ursodeoxycholyl
Ursodeoxycholyl Ester ##STR00087## m = 0-7 n = 0-7 X = NR,
7-Polycyclic Tauroursodeoxycholyl Tauroursodeoxycholyl Amide X = O,
7-Polycyclic Tauroursodeoxycholyl Tauroursodeoxycholyl Ester Cy =
Saturated or Unsaturated ring system including, any sized
Heterocycle or Carbocycles with Cic/Trans; para-, ortho- or
meta-substitution ##STR00088## m = 0-7 n = 0-7 X = NR, 7-PE UU X =
O, 7-PA UU X = O, 7,24'-PolyEther Ursodeoxychotyl Ursodeoxycholyl X
= NR, 7,24'-PolyAmine Ursodeoxycholyl Ursodeoxycholyl ##STR00089##
m = 0-7 n = 1-7 X = NR, 7-Polycyclic-Tauroursodeoxycholyl
Tauroursodeoxycholyl Amine X = O, 7-Polycyclic-Tauroursodeoxycholyl
Tauroursodeoxycholyl Ether Cy = Saturated or Unsaturated ring
system including, any sized Heterocycle or Carbocycles with
Cic/Trans; para-, ortho- or meta-substitution
TABLE-US-00003 TABLE B ##STR00090##
[0089] in embodiments: R.dbd.CH.sub.2OH, --COOM, in which M is an
alkali metal, alkaline earth metal or quaternary ammonium ion;
R.dbd.--CONHCH.sub.2CH.sub.2SO.sub.3H, R.dbd.--COOH, --COOL,
--CONH.sub.2, CH.sub.2NH.sub.2, --CONHL1 and NL1L2; L, is an alkyl,
aryl radical
[0090] In embodiments X, Y is equal to:
TABLE-US-00004 --OH, --OAc (Ac = COCH.sub.3, Acetate), --NH.sub.2,
--NHBoc (Boc = tert- --OBz (Bz = COPh, benzoyl), Butyloxycarbonyl),
--OBn (Bn = benzyl), --NHAc (Ac = COCH.sub.3, Acetate), --OTs (Ts =
Tosyl, p-toluenesulfonyl), --NHBz (Bz = COPh, benzoyl), --OMs (Ms =
methanesulfonyl), - --NHTs (Ts = Tosyl, p-toluenesulfonyl), --OTBS
(TBS = tert-Butyldimethylsilyl), --NHTf (Tf =
Trifluoromethanesulfonyl), --OTr(Tr = Triphenylmethyl), --NHMs (Ms
= methanesulfonyl), --OTf (Tf = Trifluoromethanesulfonyl),
--NBn.sub.2 (Bn = Benzyl), --OTHP (THP = Tetrahydropyran),
--N.sub.3. -Carbonyl -Alkyl
##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095##
##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100##
##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105##
##STR00106## ##STR00107## ##STR00108##
[0091] PBADs Potently and Specifically Inhibit HBV and HDV
Infection.
[0092] The inhibitory effect of some natural bile acids has been
described by us (Yan et al., J Virol, March 2014, 88(6):3273-3284)
and others: Ni et al., Gastroenterol, April 2014, 146(4):902-905;
Konig et al., J Hepatol, online 15 May 2014. Initially, we focused
on bile acid derivative modifications (Scheme 1). Primary or
secondary bile salts with or without modifications, including
cholic acid (CA), taurocholic acid (TCA), glycocholic acid (GCA),
lithocholic acid (LCA), deoxycholic acid (DCA), taurolithocholic
acid (TLCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid
(UDCA), hyodeoxycholic acid (HDCA), tauroursodeoxycholic acid
(TUDCA) and other listed derivatives were incubated with HepG2-NTCP
cells and the binding of a FITC labeled pre-S1 peptide to the
receptor was subsequently examined by fluorescence microscopy.
Consistent with the results that bile acids blocked pre-S1
lipopeptide binding to NTCP, these substrates of NTCP reduced HDV
infection as indicated by staining of intracellular HDV delta
antigen of infected cells. We further evaluated the ability of
these bile salts in inhibiting HBV infection. The levels of
secreted HBV e antigen of (HBeAg) was decreased when HepG2-NTCP
were inoculated with HBV viruses in the presence of indicated bile
salts, with TUDCA being the most potent one among all bile acid
derivatives tested. Some modified monomeric bile acids we tested,
including 7-Diz-TUDCA, Iso-TUDCA,CA-UDCA,
Diz-UDCA,3-Diz-TUDCA,Biotin-UDCA, 3-OAc-TUDCA did not show
significantly improved inhibitory efficiency over the natural
monomeric TUDCA (Table C).
TABLE-US-00005 TABLE C Monomeric Bile acids for NTCP inhibitors
##STR00109## ##STR00110## Chenodeoxycholic acid (TCDCA)
##STR00111## Tauroursodeoxycholic acid (TUDCA) ##STR00112##
Ursodeoxycholic acid (UDCA) ##STR00113## Cholic acid (CA)
##STR00114## Glycocholic acid (GCA) ##STR00115## Lithocholic acid
(LCA) ##STR00116## Deoxycholic acid (DCA) ##STR00117##
Glycochenodeoxycholic acid (GCDCA) ##STR00118##
UDCA-6-Ethyl-7-Ketone ##STR00119## UDCA-3-THP-6-Ethyl-7-Ketone
##STR00120## Glycolithocholic acid (GLCA) ##STR00121## 7-Diz-TUDCA
##STR00122## Chenodeoxycholic acid (CDCA) ##STR00123##
Hyodeoxycholic acid (HDCA) ##STR00124## Taurolithocholic acid
(TLCA) ##STR00125## Iso-tauroursodeoxycholic acid (iso-TUDCA)
##STR00126## Carbamic-UDCA (CA-UDCA) ##STR00127##
Diz-Ursodeoxycholic acid (DiZ-UDCA) ##STR00128## 3-Diz-TUDCA
##STR00129## Biotin-UDCA ##STR00130## 3-OAc-TUDCA ##STR00131##
Trideoxylcholic acid (TDCA) ##STR00132## Taurocholic acid (TCA)
##STR00133## UDCA-6-Ethyl ##STR00134## UDCA-3-iso-Ms ##STR00135##
UDCA-3-N.sub.3 ##STR00136## UDCA-3-NH.sub.2 ##STR00137##
UDCA-3-TBS-7-Ms ##STR00138## UDCA-5,6-Olefin ##STR00139##
UDCA-6,7-Epoxy ##STR00140## TCDCA-6(S)-OH ##STR00141## UDCA-3-TBS
##STR00142## TUDCA-3-NH.sub.2 ##STR00143## TUDCA-3-N.sub.3
##STR00144## TUDCA-6S-OH ##STR00145## TUDCA-3-Biotin ##STR00146##
TUDCA-7-Biotin ##STR00147## TUDCA-3,7-F ##STR00148## T-estrol
[0093] Based on quantitative analysis of active impurities we
examined several polymeric bile acid derivatives (Table D). We
first synthesized the "head-to-tail" polymers, such as dimeric 3-UT
(NQL-012) and 7-UT(NQL-015). For "head-to-head" motif, we tried
DIU(NQL-009), which was connected by ester bond directly, other
type of connections are under studied. For "tail-to-tail"
connection, 3-TPT (NQL-018) and 3-TPAT (NQL-044) was linked
together through double ester or amide bonds with 5 carbons in the
middle, more or less bond connection will be studied as soon as
possible. We found 3-UT(NQL-012), 7-UT(NQL-015), DIU(NQL-009),
3-TPT(NQL-018) and 3-TPAT (NQL-044) have high antiviral activity
against both HBV and HDV, with an IC50 of the 3-TPT for HBV down to
51 nM (FIG. 5, please also see FIGS. 2 and 3).
TABLE-US-00006 TABLE D Molecular design of tested PBADs
##STR00149##
[0094] These data indicate that dimeric bile acids based on UDCA
and TUDCA motif are active against HBV and HDV infection.
[0095] The invention encompasses all combinations of two same or
different monomeric bile acid derivatives (formula I-IV) disclosed
herein, including alternative linkers, their pharmaceutically
tolerated salts, conjugated acid form, metabolic derivatives, and
the synthesis, purification and the use of these derivatives and
salts, including the structures shown as 3-UT (NQL-012), 7-UT
(NQL-015), DIU (NQL-009), 3-TPT and 3-TPAT (NQL-044), which has two
UDCAs or TUDCAs connected with ester bond between acid group on one
UDCA or TUDCA and one of hydroxyl groups on another UDCA or TUDCA
(Scheme 2), and including dimeric bile acids derivatives of the
formula (II), (III), (IV) and (V) in Scheme 3.
[0096] In the Table E the U and T in the dimeric bile acid
derivatives can be symmetric (identical to each other) or
asymmetric (combination of UDCA and TUDCA). The L is the conjugated
linkage group between different position of U and T. For the
"head-to-head" formula (V) U1-L-U2, L connected two side chains of
U1 and U2 at positions (via the carbon atom or the substituent
thereon) of 20 to 24 via covalent bond. For the "head-to-tail"
formula (VI) 3-U1-L-U2, L connected to the position (via the carbon
atom or the substituent thereon) of 20 to 24 on one side chain of
U1 and 3-positions (via the carbon atom or the substituent X atom)
of U2 via covalent bond. For the "head-to-tail" formula (VII)
7-U1-L-U2, L connected to the position (via the carbon atom or the
substituent thereon) of 20 to 24 on one side chain of U1 and
7-positions (via the carbon atom or the substituent X atom) of U2
via covalent bond. For the "tail-to-tail" formula (VIII) T1-L-T2, L
connected to 3- or 7-position (via the carbon atom or the
substituent X or Y atoms) of T1 and 3- or 7-position (via the
carbon atom or the substituent X or Y atoms) of T2 via covalent
bond. The number n and m can be any Arabic numerals.
TABLE-US-00007 TABLE E Chemical design for dimeric bile acids
derivatives based on UDCA ##STR00150##
[0097] The invention encompasses dimeric bile acids based on
monomeric bile acid motif of B (Table F), and in addition to the
UDCA and TUDCA dimeric derivatives showed in the Table E, the
invention encompasses generally dimeric bile acids derivatives of
formula (IX), (X) and (XI) in Table F.
[0098] In the table F: B1 and B2 in the dimeric bile acid
derivatives can be symmetric (identical to each other) or
asymmetric (combination of all the above mentioned monomeric bile
acid derivative possibilities). L is the conjugated linkage group
between different position of B1 and B2. For the "head-to-head"
formula (IX) B1-L-B2, L connected two side chains of B1 and B2 at
positions (the carbon atom or the substituent thereon) of 20 to 24
via covalent bond. For the "head-to-tail" formula (X) B1-L-B2, L
connected to the position (via the carbon atom or the substituent
atoms thereon) of 20 to 24 on one side chain of B1 and any
positions (via the carbon atom or the substituent atoms thereon, on
the rings of A, B, C and D) of 1 to 19 of B2 via covalent bond. For
the "tail-to-tail" formula (XI) B1-L-B2, L connected to any one
position (via the carbon atom or the substituent atoms thereon, on
the rings of A, B, C and D) of 1 to 19 of B1 and any positions (via
the carbon atom or the substituent atoms thereon, on the rings of
A, B, C and D) of 1 to 19 of B2 via covalent bond. The number n and
m can be any Arabic numerals.
TABLE-US-00008 TABLE F Chemical design for dimeric bile acids
derivatives ##STR00151##
[0099] We also tested and found anti-HBV and anti-HDV activity for
trimeric bile acid derivatives, though the activity of linear
trimeric bile acid 3UUT (see table D) was not as high as the
dimeric counterparts (FIG. 5, also see FIGS. 2 and 3). Active
trimmers are encompassed by formula (XII) in the Table G.
TABLE-US-00009 TABLE G Chemical design for trimeric bile acid
derivatives. ##STR00152##
[0100] For the trimeric structure (XII) in Table G:
[0101] L is the conjugated linkage group between B1, B2 and B3.
Wherein B1, B2 and B3 are monomeric bile acid derivatives. For the
trimeric formula (XII), L is connected to the monomeric bile acid
derivatives at any positions (the carbon atom or the substituent
atoms thereon) of 1 to 24 via covalent bond. The number n, m and o
can be any Arabic numerals.
[0102] PBADs Inhibit HBV Infection at the Entry Level.
[0103] To dissect the anti-viral mechanism of PBADs, we conducted
time course studies to find their action time windows. We found
these PBADs were active when added before or during viral
inoculation on the cells, but not post viral inoculation,
indicating they may act on the early entry process (FIG. 3).
[0104] PBADs Significantly Block Substrate Uptake of NTCP.
[0105] We further analyzed the influence of PBAD treatment on NTCP
mediated [3H] labeled taurocholate uptake (FIG. 4). In the
co-incubation assay, most of the PBADs such as 3-UT (NQL-012), 7-UT
(NQL-015) and 3TPT (NQL-018) that efficiently inhibited HBV and HDV
infection also inhibited NTCP substrate uptake, with a
significantly higher efficiency than that of TUDCA (FIG. 4A,B). We
also evaluated the uptake inhibition of 3UT(NQL-012) and
3TPT(NQL-018) in the pretreatment assay. The results showed the
IC50 of 3UT (NQL-012) is 108.6 nM, 3TPT(NQL-018) is 58.7 nM, close
to IC50 of a positive control reagent (preS1 peptide of HBV L
protein) (FIG. 4C).
[0106] The IC50 of PBADs of HBV Infection on HepG2-NTCP Cells.
[0107] We conducted dose dependence inhibition assay to determine
more quantitatively the IC50 of the PBADs. As illustrated in FIG.
5, the 3TPT(NQL-018) showed the best anti-HBV activity, with an
IC50 of 51.04 nM, about 100 times lower than that of its monomer
precursor (TUDCA). The potency of 3UT(NQL-012) and DiU(NQL-009) was
slightly lower than 3TPT(NQL-018). Their IC50 values were 100 nM
and 110 nM, respectively.
[0108] In Vitro Cytotoxicity of PBADs.
[0109] We also conducted experiments to determine the LD50 on
HepG2-NTCP cell line of the three PBADs (3-UT (NQL-012), DIU
(NQL-009), 3TPT (NQL-018)). We treated cells by different
concentrations of PBADs for 48 hrs and captured the images of cells
after they were further cultured for 3 days in PMM. As shown in
FIG. 6, treatment by 3UT (NQL-012) led to significant cell death at
high concentration, with a LD50 near 25 .mu.M. No detectable
cytotoxicity was observed upon treatment of DIU(NQL-009) and
3TPT(NQL-018) up to 200 .mu.M, which is 2000-4000 folds higher than
its inhibitory concentration for HBV infection.
[0110] PBADs Inhibit Viral Infection by Targeting NTCP and Block
its Interaction with HBV preS1 Domain.
[0111] We employed FITC-preS1 peptide binding assay to examine
whether PBADs block FITC-preS1 binding to NTCP. As shown in FIG.
7A, some PBADs indeed blocked the interaction between preS1 and
NTCP, and the efficiency was correlated to their anti-HBV activity.
Interestingly, high concentration of 3UUT(NQL-016), DIU(NQL-009)
and CSA led to the aggregation of FITC-preS1 peptide, which may be
attributed to their hydrophobic properties. Furthermore, we tested
the effect of pretreatment versus co-incubation of 3TPT(NQL-018) at
different concentrations. The FIG. 7B showed that the effect of
pretreatment of 3TPT(NQL-018) was not as strong as that of
co-incubation.
[0112] The In Vitro Stability of PBADs could PBADs could be
Enhanced by Optimization of the Linkage Bond.
[0113] We conducted stability analysis of several PBADs with
similar structure but of different linkage. FIG. 10 showed that the
ester bond based 3TPT(NQL018, table H) is not stable upon long time
incubation with the inoculum. In comparison, NQL044 and NQL055 are
much more stable as no significant potency decrease was observed.
This indicates the in vitro and in vivo stability is to a large
extent determined by the feature of the linkage groups.
TABLE-US-00010 TABLE H Chemical structural examples of dimeric bile
acids derivatives of UDCA with different linkages. ##STR00153##
NIBS-Qi-WHLi-018 NQL-018(3-TPT) ##STR00154## NIBS-Qi-WHLi-019
NQL-019 ##STR00155## NIBS-Qi-WHLi-020 NQL-020 ##STR00156##
NIBS-Qi-WHLi-021 NQL-021 ##STR00157## NIBS-Qi-WHLi-022 NQL-022
##STR00158## NIBS-Qi-WHLi-023 NQL-023 ##STR00159## NIBS-Qi-WHLi-024
NQL-024 ##STR00160## NIBS-Qi-WHLi-025 NQL-025 ##STR00161##
NIBS-Qi-WHLi-026 NQL-026 ##STR00162## NIBS-Qi-WHLi-027 NQL-027
##STR00163## NIBS-Qi-WHLi-028 NQL-028 ##STR00164## NIBS-Qi-WHLi-029
NQL-029 ##STR00165## NIBS-Qi-WHLi-030 NQL-030 TCDCA-TCDCA
##STR00166## NIBS-Qi-WHLi-032 NQL-032 TLCA-TLCA ##STR00167##
NIBS-Qi-WHLi-043 NQL-043 ##STR00168## NIBS-Qi-WHLi-044 NQL-044
##STR00169## NQL-048 ##STR00170## NQL-052 ##STR00171## NQL-053
##STR00172## NQL-054 ##STR00173## NQL-055 ##STR00174## NQL-056
##STR00175## NQL-057 ##STR00176## NQL-058 ##STR00177## NQL-059
##STR00178## NQL-060
[0114] The Antiviral Potency of PBADs is Also Repeatable on Other
HBV Infection System (HepaRG) with Similar Inhibition Profile.
[0115] As showed in FIG. 1, we conducted HBV infection assay on
HepaRG cells, in which endogenous NTCP expression could be induced
upon DMSO. HBV infection was conducted in the presence of NQL028,
NQL018, NQL029, NQL019 and NQL021, all of which were bis-ester
based PBADs with different linkage length. All of them inhibited
HBV infection and the inhibition profile is similar to the result
from HBV infection on HepG2-NTCP cells.
[0116] Structure Activity Relationship Analysis of the PBADs.
[0117] In FIG. 12, we showed the results of HepG2-NTCP cells
infected with HBV in the presence of the indicated PBADs across a
variety of monomers and linkages with different concentration. Each
figure compared the efficacy of different PBADs (Table H) with
similar structure, and the influence of their structure difference
to antiviral potency was analyzed according to the results.
[0118] Materials and Methods.
[0119] Cell culture. Human hepatocarcinoma cell line HepG2 was from
American Type Culture Collection (ATCC); human hepatocarcinomacell
line (Huh-7) was from the Cell Bank of Type Culture Collection,
Chinese Academy of Sciences. They were cultured with Dulbecco's
Modification of Eagle's Medium (DMEM; Invitrogen) supplemented with
10% fetal bovine serum (FBS, Gibco) at 37.degree. C. with regular
passage of every 2 days. HepG2NTCP stable cell line was generated
from HepG2 cells and maintained in DMEM supplemented with 10% FBS
and 500 .mu.g/ml G418. HepG2-NTCP stable cell line were cultured on
collagen (BD) coated plates or dishes. These cells were cultured in
PTH maintenance medium (PMM) for 24 hrs before viral infection,
peptide binding, substrate uptake, and other NTCP related
experiments.
[0120] Peptide, Antibodies and Other Reagents.
[0121] FITC-pre-S1 peptide is derived from pre-S1 domain of HBV
(C-type, GenBank accession no. EU554535.1) containing the first 59
residues, with an amino-terminal myristoylation modification and a
carboxyl-terminal fluorescein isothiocyanate (FITC) conjugation;
myr(+)47 peptide containing 2-47 residues of pre-S1 domain of HBV
with N-terminal myristoylation. All these peptides were synthesis
by SunLight peptides Inc. (Beijing, China). 1C10 is a mouse
monoclonal antibody (mAb) recognizing the HBV core protein; 4G5 is
a mouse mAb specifically targeting HDV delta antigen, #36 antibody
is a mouse mAb specifically targeting NTCP. All mAbs were developed
by the conventional hybridoma technology in the lab. Most natural
bile acids are purchased form Sigma Aldrich. [3H] labeled
taurocholate with activity of 15.3 Ci/mmol (0.185 TBq/mmol) and
liquid scintillation cocktail (Ultima GOLD.TM. XR) were purchased
from Perkin Elmer.
[0122] Virus Production.
[0123] Virions were produced from Huh7 cells after transfection of
viral production plasmids. After transfection, cells were
replenished with PMM, and virus containing medium were collected at
3 days and 6 days post transfection, centrifuged and stored at
-80.degree. C.
[0124] FITC-preS1 Peptide Binding Assay.
[0125] NTCP expressing cells were cultured in PTH maintenance
medium (PMM) for 24 hrs before conducting this assay. For
Immunofluorescence microscopy, cells were incubated with 400 nM
FITC-pre-S1 peptide diluted in WME or PMM at 37.degree. C. for
about 2-3 hrs. Subsequently, cells were washed once by WME, and
then directly visualized with a Fluorescence Microscope.
[0126] HBV and HDV Infection Assay.
[0127] HepG2NTCP Cells were cultured in PMM for 12-24 hrs before
infection, and then inoculated with 200 multiplicities of genome
equivalents (mge) of HBV, or 500 mge of HDV in the presence of 5%
PEG8000 in PMM at 37.degree. C. for about 24 hrs. Chemicals to be
tested were added to the cells as indicated in the specific assays.
The inoculum was replenished by PMM post infection. The infections
were detected at day 5 post infection (dpi).
[0128] [3H] Substrate Uptake Assay.
[0129] [3H]taurocholate uptake assay was conducted following a
protocol as previously described(60). In general, HepG2-NTCP cells
were cultured in PMM for 12-24 hrs, then were treated with or
without indicated chemicals before uptake assay. For substrate
uptake assay, cells were generally incubated with 0.5 .mu.l (0.5
.mu.Ci) [3H]-taurocholate dissolved in Na+ Ringer's solutions for
10 mins at 37.degree. C. with or without the presence of chemicals.
Subsequently, cells were washed once by PBS and lysed by 100 .mu.l
of 1% TritonX-100 in H.sub.2O for 5 mins at room temperature. The
lysate was transferred into liquid scintillation tube and mixed
with 900 .mu.l liquid scintillation cocktail (Ultima GOLD.TM. XR)
(Perkin Elmer, USA). Liquid scintillation counting was performed on
a Perkin Elmer 1450 LSC Liquid Scintillation Counter and
Luminescence Counter.
[0130] ELISA Assay for the Detection of HBeAg and HBsAg.
[0131] ELISA kits for HBeAg and HBsAg detection were from Wantai
Pharm Inc. (Beijing, China). Supernatant from infected HepG2NTCP
cells was collected on 2-5 days post infection. The secreted HBeAg
and HBsAg in the culture medium was measured with a commercial kit
from Wantai Pharm Inc (Beijing, China) by following the
manufactory's instructions.
[0132] Immunostaining Assay.
[0133] For HBV infection, infected cells were washed twice with PBS
and fixed in 3.7% paraformaldehyde (PFA) at room temperature for 10
mins. Subsequently, cells were permeabilized with 0.5% Trition
X-100/PBS for 10 mins at room temperature, blocked with 3% BSA at
37.degree. C. for 1 hour, and followed by incubating with 5
.mu.g/ml mouse mAb1C10 which recognizes HBcAg, and followed by
staining with FITC-conjugated secondary antibody. Nucleus was
stained with DAPI in blue. The stained cells were imaged with the
fluorescence microscope (Nikon). For HDV infection, on 5 dpi, HDV
infected cells were fixed with 100% methanol at room temperature
for 10 mins, intracellular delta antigens were then stained with 5
m/ml of FITC conjugated 4G5 and nucleus were stained with DAPI in
blue. Images were collected by an Eclipse Ti Fluorescence
Microscope (Nikon) and a representative picture is shown.
[0134] Synthesis of PBADs.
[0135] Preparing compounds of the general formula (I) to (VIII)
from B1 or B2 by known or if not known, by the processes described
below in details. The linkage L being generated from activated
functional group on either B1 or B2 by construction of a covalent
bond, in particular in the course of condensation or nucleophilic
substitution reactions. Unprotective strategies were applied
through the synthesis of 3-substituted or 3-linked polymeric bile
acids. For example, condensation and substitution on bile acids is
preferably happened in position 3, then position 7. Activated
carboxylic acids, such as acid chlorides, anhydrides or aminoester
will react directly with free alcohol or amino group to form ester
or amide bond in the presence of organic or inorganic bases such as
trialkylamine, pyridines, NaOH, KOH and so on. Suitable solvents
for this type of reaction include tetrahydrofuran, methylene
chloride, ether, acetonitile, dimethoxylethane and
dimethylformamide. The reactions on the position of 7 and other
positions can be achieved selectively by the use of suitable
protection groups such as acetyl ester, trityl, alkyl(aryl)silyl,
benzyl, allyl carbonate, Carboxybenzyl and ethers.
[0136] The preparation of bioactive polymeric bile acid derivatives
were illustrated in the table C and D as exemplified by synthesis
of 3-UT (NQL-012), 3-TPT (NQL-018) and 3-TAPT (NQL-044) from
ursodeoxycholic acid (UDCA).
TABLE-US-00011 TABLE I The synthetic route to 3-UT (NQL-012)
##STR00179## ##STR00180##
[0137] In a 10 mL, one-necked, round-bottomed flask,
(4R)-4-((3R,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadec-
ahydro-1H-cyclopenta [.alpha.]phenanthren-17-yl) pentanoic acid
(UDCA) (23.5 mg, 0.06 mmol, 1.5 equiv) was dissolved in DMF (1 mL)
at room temperature. Then
2-((4R)-4-((3R,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexa-
decahydro-1H-cyclopenta[.alpha.]phenanthren-1'7-yl) pentanamido)
ethanesulfonic acid (TUDCA) (20 mg, 0.04 mmol, 1.0 equiv),
N,N-Diisopropylethylamine (DIEA) (15.5 mg, 0.12 mmol, 3.0equiv),
4-Dimethylaminopyridine (DMAP) (5 mg, 0.04 mmol, 1.0equiv) and
N,N'-Dicyclohexylcarbodiimide (DCC) (16.5 mg, 0.08 mmol, 2.0equiv)
was added. After the addition was complete, the mixture was stirred
at room temperature for 14 h. The mixture was concentrated and
purified by preparative-HPLC to obtain target product as a white
solid (5.0 mg).
[0138] The examples of Table J were obtained in analogy to Table
I
TABLE-US-00012 Examples ##STR00181## NQL-012
C.sub.50H.sub.83NO.sub.9S MW: 874 [M - H].sup.-: 873 ##STR00182##
NQL-015 C.sub.50H.sub.85NO.sub.8S MW: 860 [M - H].sup.-: 859
##STR00183## NQL-016 C.sub.74H.sub.121NO.sub.12S MW: 1248 [M -
H].sup.-: 1247 ##STR00184## NQL-017 C.sub.50H.sub.83NO.sub.9S MW:
874 [M - H].sup.-: 873 ##STR00185## NQL-031
C.sub.50H.sub.83NO.sub.9S MW: 874 [M - H].sup.-: 873 ##STR00186##
NQL-057 C.sub.36H.sub.59N.sub.3O.sub.8S.sub.2 MW: 726 [M -
H].sup.-: 725
TABLE-US-00013 TABLE K The synthetic route to NQL-018 ##STR00187##
##STR00188## ##STR00189##
[0139] In a 10 mL, one-necked, round-bottomed flask,
2-((4R)-4-((3R,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexa-
decahydro-1H-cyclopenta[.alpha.]phenanthren-17-yl) pentanamido)
ethanesulfonic acid (UDCA) (50.0 mg, 0.1 mmol, 2.0equiv) was
dissolved in DMF (1 mL) at room temperature. Then glutaric acid
(6.6 mg, 0.05 mmol, 1.0 equiv), N,N-Diisopropylethylamine (DIEA)
(25.8 mg, 0.2 mmol, 4.0equiv), 4-Dimethylaminopyridine (DMAP) (6.1
mg, 0.05 mmol, 1.0equiv) and
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (23.0 mg, 0.12
mmol, 2.4equiv) was added. After the addition was complete, the
mixture was stirred at room temperature for 14 h. The mixture was
concentrated and used directly.
[0140] In a 10 mL, one-necked, round-bottomed flask, the above
5-(((3R,7 S,9 S,10 S,13R,14
S,17R)-7-hydroxy-10,13-dimethyl-17-((R)-5-oxo-5-((2-sulfoethyl)amino)pent-
an-2-yl)hexadecahydro-1H-cyclopenta[.alpha.]phenanthren-3-yl)oxy)-5-oxopen-
tanoic acid (30.6 mg, 0.05 mmol, 1.0equiv) was dissolved in DMF (1
mL) at room temperature. Then
2-((4R)-4-((3R,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexa-
decahydro-1H-cyclopenta[.alpha.]phen-anthren-17-yl)pentanamido)
ethanesulfonic acid (25.0 mg, 0.05 mmol, 1.0equiv),
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate (HATU) (19.0 mg, 0.05 mmol, 1.0equiv)
and N,N-Diisopropylethylamine (DIEA) (25.8 mg, 0.2 mmol, 4.0equiv)
were added. After the addition was complete, the mixture was
stirred at room temperature for 14 h. The mixture was concentrated
and purified by pre-HPLC to get target product as a white solid
(9.3 mg), which was confirmed by NMR and Mass spectrum.
[0141] The examples of Table L were obtained in analogy to Table
K.
TABLE-US-00014 Examples ##STR00190## NQL-018
C.sub.57H.sub.94N.sub.2O.sub.14S.sub.2 MW: 1095 [M - H].sup.-: 1095
##STR00191## NQL-019 C.sub.59H.sub.98N.sub.2O.sub.14S.sub.2 MW:
1123 [M - H].sup.-: 1122 ##STR00192## NQL-028
C.sub.56H.sub.92N.sub.2O.sub.14S.sub.2 MW: 1081 [M - H].sup.-: 1080
##STR00193## NQL-029 C.sub.58H.sub.96N.sub.2O.sub.14S.sub.2 MW:
1109 [M - H].sup.-: 1108 ##STR00194## NQL-030
C.sub.57H.sub.94N.sub.2O.sub.14S.sub.2 MW: 1095 [M - H].sup.-: 1094
##STR00195## NQL-032 C.sub.57H.sub.94N.sub.2O.sub.12S.sub.2 MW:
1063 [M - H].sup.-: 1062
TABLE-US-00015 TABLE M The synthetic route to NQL-044 ##STR00196##
##STR00197## ##STR00198## ##STR00199##
[0142] In a 100 mL, one-necked, round-bottom flask,
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[.alpha.]phenanthren-1'7-yl) pentanoic acid (10.0
g, 25.5 mmol, 1.0 equiv) was dissolved in MeOH (100 mL) at room
temperature. Then PTSA (cat.) was added. After the addition was
complete, the mixture was stirred at 80.degree. C. for 14 h. The
mixture was concentrated and basified with con.NaHCO.sub.3 to
pH>8, then extracted with EA, concentrated under reduced
pressure to get target product (9.3 g) which was confirmed by
NMR.
[0143] In a 100 mL, three-necked, round-bottom flask, PPh3 (2.6 g,
10.0 mmol, 2.0 equiv) was dissolved in anhydrous THF (100 mL) at
-15.degree. C., then DIAD (2.02 g, 10 mmol, 2.0 equiv) was added
under Ar dropwise. After 10 min, methyl
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[.alpha.]phenanthren-17-yl) pentanoate (2.0 g, 5.0
mmol, 1.0 equiv) dissolved in THF (10 mL) was added dropwise. The
mixture was stirred at room temperature for 30 min. Then
methanesulfonic acid (960 mg, 10.0 mmol, 2.0 equiv) was added.
After the addition was complete, the mixture was stirred at room
temperature for 14 h. The mixture was quenched by the addition of
con.NaHCO3 to pH>8 and extracted with EA, concentrated under
reduced pressure and purified by silica column chromatography
(PE:EA=1:1) to get target product (0.6 g) which was confirmed by
NMR.
[0144] In a 25 mL, one-necked, round-bottom flask, methyl
(4R)-4-((3S,7S,9S,10S,13R,14S,17R)-7-hydroxy-10,13-dimethyl-3-((methylsul-
fonyl)oxy)hexadecahydro-1H-cyclopenta[.alpha.]phenanthren-17-yl)
pentanoate (1.2 g, 2.5 mmol, 1.0 equiv) was dissolved in DMF (10
mL) at room temperature. Then NaN3 (325 mg, 5.0 mmol, 2.0 equiv)
was added. After the addition was complete, the mixture was stirred
at 80.degree. C. for 1.5 h. The mixture was washed with water and
extracted with EA, concentrated and used directly.
[0145] The crude product was dissolved in MeOH (10 mL) at room
temperature. Then Pd/C (0.5 g) was added and bubbled with a balloon
full of H2. After the addition was complete, the mixture was
stirred at room temperature for 14 h under H2. The mixture was
concentrated and purified by chromatography column (DCM: MeOH=5:1)
to get target product as a yellow solid (700 mg), which was
confirmed by NMR and UPLC/MS.
[0146] In a 10 mL, one-necked, round-bottom flask, methyl
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3-amino-7-hydroxy-10,13-dimethylhexade-
cahydro-1H-cyclopenta[.alpha.]phenanthren-17-yl) pentanoate (120.0
mg, 0.3 mmol, 1.0 equiv) was dissolved in DMF (2.0 mL) at room
temperature. Then 2-glutaric acid (39.6 mg, 0.3 mmol, 1.0 equiv),
DIEA (116.0 mg, 0.9 mmol, 3.0 equiv), DMAP (36 mg, 0.3 mmol, 1.0
equiv) and EDC (173 mg, 0.9 mmol, 3.0 equiv) was added. After the
addition was complete, the mixture was stirred at room temperature
for 24 h. The mixture was confirmed by UPLC, concentrated and
purified by chromatography column (DCM: MeOH=20:1) to get target
product (52 mg).
[0147] In a 10 mL, one-necked, round-bottom flask, methyl
(4R)-4-((3S,7R,10S,13R,17R)-7-hydroxy-3-(5-(((3R,7S,10S,13R,17R)-7-hydrox-
y-17-((R)-5-methoxy-5-oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclo-
penta[.alpha.]phenanthren-3-yl)amino)-5-oxopentanamido)-10,13-dimethylhexa-
decahydro-1H-cyclopenta[.alpha.]phenanthren-17-yl) pentanoate (52.0
mg, 0.057 mmol, 1.0 equiv) was dissolved in THF (1.0 mL)/MeOH (0.2
mL)/H2O (1.0 mL) at room temperature. Then LiOH (7.2 mg, 0.17 mmol,
3.0 equiv) was added and stirred at room temperature for 4 h. The
mixture was acidified with 1N HCl to pH<3 and extracted with EA,
concentrated and used directly.
[0148] The crude product was dissolved in DMF (1.5 mL) at room
temperature. Then 2-aminoethanesulfonic acid (42.5 mg, 0.34 mmol,
6.0 equiv), DIEA (58.8 mg, 0.46 mmol, 8.0 equiv), DMAP (7 mg, 0.05
mmol, 1.0 equiv) and EDC (32.6 mg, 0.17 mmol, 3.0 equiv) was added.
After the addition was complete, the mixture was stirred at room
temperature for 14 h. The mixture was concentrated and purified by
pre-HPLC to get target product as a white solid (0.7 mg), which was
confirmed by NMR and Mass spectrum.
[0149] The examples of Table N were obtained in analogy to Table
M
TABLE-US-00016 Examples ##STR00200## NQL-020
C.sub.53H.sub.86N.sub.2O.sub.8 MW: 879 [M - H].sup.-: 878
##STR00201## NQL-021 C.sub.57H.sub.96N.sub.4O.sub.12S.sub.2 MW:
1093 [M - H].sup.-: 1092 ##STR00202## NQL-022
C.sub.55H.sub.92N.sub.4O.sub.12S.sub.2 MW: 1065 [M - H].sup.-: 1064
##STR00203## NQL-043 C.sub.55H.sub.91N.sub.3O.sub.10S MW: 986 [M -
H].sup.-: 985 ##STR00204## NQL-044
C.sub.57H.sub.96N.sub.4O.sub.12S.sub.2 MW: 1094 [M - H].sup.-: 1093
##STR00205## NQL-060 C.sub.57H.sub.94N.sub.6O.sub.11S.sub.2 MW:
1104 [M - H].sup.-: 1103
TABLE-US-00017 TABLE O The synthetic route to NQL-055 ##STR00206##
##STR00207## ##STR00208## ##STR00209## ##STR00210##
[0150] In a 100 mL, one-necked, round-bottom flask,
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[.alpha.]phenanthren-1'7-yl) pentanoic acid (406.0
mg, 1.0 mmol, 1.0 equiv) and 3-bromoprop-1-yne (476.0 mg, 4.0 mmol,
4.0 equiv) was dissolved in DMF (2 mL) at room temperature. After
the addition was complete, the mixture was stirred at room
temperature for 14 h. The mixture was washed with water and
extracted with EA, concentrated and purified by chromatography
column (DCM:MeOH=20:1) to get target product (50 mg) which was
confirmed by NMR.
[0151] In a 100 mL, one-necked, round-bottom flask,
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[.alpha.]phenanthren-17-yl) pentanoic acid (10.0
g, 25.5 mmol, 1.0 equiv) was dissolved in MeOH (100 mL) at room
temperature. Then PTSA (cat.) was added. After the addition was
complete, the mixture was stirred at 80.degree. C. for 14 h. The
mixture was concentrated and basified with con.NaHCO.sub.3 to
pH>8, then extracted with EA, concentrated under reduced
pressure to get target product (9.3 g) which was confirmed by
NMR.
[0152] In a 100 mL, three-necked, round-bottom flask, PPh.sub.3
(2.6 g, 10.0 mmol, 2.0 equiv) was dissolved in anhydrous THF (100
mL) at -15.degree. C., then DIAD (2.02 g, 10 mmol, 2.0 equiv) was
added under Ar dropwise. After 10 min, methyl
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[.alpha.]phenanthren-17-yl) pentanoate (2.0 g, 5.0
mmol, 1.0 equiv) dissolved in THF (10 mL) was added dropwise. The
mixture was stirred at room temperature for 30 min. Then
methanesulfonic acid (960 mg, 10.0 mmol, 2.0 equiv) was added.
After the addition was complete, the mixture was stirred at room
temperature for 14 h. The mixture was quenched by the addition of
con.NaHCO.sub.3 to pH>8 and extracted with EA, concentrated
under reduced pressure and purified by silica column chromatography
(PE:EA=1:1) to get target product (0.6 g) which was confirmed by
NMR.
[0153] In a 25 mL, one-necked, round-bottom flask, methyl
(4R)-4-((3S,7S,9S,10S,13R,14S,17R)-7-hydroxy-10,13-dimethyl-3-((methylsul-
fonyl)oxy)hexadecahydro-1H-cyclopenta[.alpha.]phenanthren-17-yl)
pentanoate (1.2 g, 2.5 mmol, 1.0 equiv) was dissolved in DMF (10
mL) at room temperature. Then NaN.sub.3 (325 mg, 5.0 mmol, 2.0
equiv) was added. After the addition was complete, the mixture was
stirred at 80.degree. C. for 1.5 h. The mixture was washed with
water and extracted with EA, concentrated purified by
chromatography column (PE:EA=5:1) to get target product as a yellow
solid (700 mg), which was confirmed by NMR.
[0154] In a 10 mL, one-necked, round-bottom flask, methyl
(4R)-4-((3R,7S,95 S,13R,14
S,17R)-3-azido-7-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[.alpha-
.]phenanthren-17-yl) pentanoate (35.5 mg, 0.084 mmol, 1.5 equiv)
and methyl
(4R)-4-((3R,75,95,10S,13R,14S,17R)-3-(ethynyloxy)-7-hydroxy-10,13--
dimethylhexadecahydro-1H-cyclopenta[.alpha.]phenanthren-17-yl)
pentanoate (25.0 mg, 0.056 mmol, 1.0 equiv) was dissolved in DMF
(2.0 mL) at room temperature. Then CuSO.sub.4.5H.sub.2O (15.12 mg,
0.056 mmol, 1.0 equiv) and Sodium L-ascorbate (22.0 mg, 0.112 mmol,
2.0 equiv) in H.sub.2O (2.0 mL) was added. After the addition was
complete, the mixture was stirred at room temperature for 24 h. The
mixture was washed with water and extracted with DCM, concentrated
and purified by chromatography column (DCM: MeOH=20:1) to get
target product (20 mg).
[0155] In a 10 mL, one-necked, round-bottom flask, methyl
(4S)-4-((3S,7R,9R,10R,13S,14R,17S)-7-hydroxy-3-((1-((3R,7S,9S,10S,13R,14S-
,17R)-7-hydroxy-17-((R)-5-methoxy-5-oxopentan-2-yl)-10,13-dimethylhexadeca-
hydro-1H-cyclopenta[.alpha.]phenanthren-3-yl)-1H-1,2,3-triazol-4-yl)methox-
y)-10,13-dimethylhexadecahydro-1H-cyclopenta[.alpha.]phenanthren-17-yl)
pentanoate (20.0 mg, 0.023 mmol, 1.0 equiv) was dissolved in THF
(1.0 mL)/MeOH (0.2 mL)/H.sub.2O (1.0 mL) at room temperature. Then
LiOH (4.2 mg, 0.114 mmol, 5.0 equiv) was added and stirred at room
temperature for 4 h. The mixture was acidified with 1N HCl to
pH<3 and extracted with EA, concentrated and used directly.
[0156] The crude product was dissolved in DMF (1.5 mL) at room
temperature. Then 2-aminoethanesulfonic acid (17.8 mg, 0.142 mmol,
6.0 equiv), DIEA (24.7 mg, 0.192 mmol, 8.0 equiv), DMAP (3 mg,
0.024 mmol, 1.0 equiv) and EDC (13.6 mg, 0.71 mmol, 3.0 equiv) was
added. After the addition was complete, the mixture was stirred at
room temperature for 14 h. The mixture was concentrated and
purified by pre-HPLC to get target product as a white solid (4.4
mg), which was confirmed by NMR.
[0157] The examples of Table P were obtained in analogy to Table
O.
TABLE-US-00018 Examples ##STR00211## NQL-055
C.sub.55H.sub.91N.sub.5O.sub.11S.sub.2 MW: 1062 [M - H].sup.-:
1061
TABLE-US-00019 TABLE Q The synthetic route to NQL-052 ##STR00212##
##STR00213## ##STR00214##
[0158] In a 100 mL, one-necked, round-bottom flask,
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[.alpha.]phenanthren-1'7-yl) pentanoic acid (10.0
g, 25.5 mmol, 1.0 equiv) was dissolved in MeOH (100 mL) at room
temperature. Then PTSA (cat.) was added. After the addition was
complete, the mixture was stirred at 80.degree. C. for 14 h. The
mixture was concentrated and basified with con.NaHCO.sub.3 to
pH>8, then extracted with EA, concentrated under reduced
pressure to get target product (9.3 g) which was confirmed by NMR
and MS spectrum.
[0159] In a 25 mL, one-necked, round-bottom flask, methyl methyl
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[.alpha.]phenanthren-17-yl) pentanoate (203.0 mg,
0.5 mmol, 1.0 equiv) was dissolved in DMF (10 mL) at room
temperature. Then 1,5-dibromopentane (111.5 mg, 0.5 mmol, 1.0
equiv) and KOH (56.0 mg, 1.0 mmol, 2.0 equiv) was added. After the
addition was complete, the mixture was stirred at room temperature
for 36 h. The mixture was washed with water and extracted with EA,
concentrated to get a crude product which was used directly.
[0160] In a 10 mL, one-necked, round-bottom flask, dimethyl
4,4'-((3R,3'R,7S,7'S,10S,10'S,13R,13'R,17R,17'R)-(pentane-1,5-diylbis
(oxy)) bis
(7-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[.alpha.]phenanthrene-
-3,17-diyl)) (4R,4'R)-dipentanoate (crude) was dissolved in THF
(1.0 mL)/MeOH (0.2 mL)/H.sub.2O (1.0 mL) at room temperature. Then
LiOH (7.2 mg, 0.17 mmol, 3.0 equiv) was added and stirred at room
temperature for 4 h. The mixture was acidified with 1N HCl to
pH<3 and extracted with EA, concentrated purified by
chromatography column (DCM: MeOH=20:1) to get target product (47
mg) which was confirmed by UPLC/MS.
[0161] The product was dissolved in DMF (1.5 mL) at room
temperature. Then 2-aminoethanesulfonic acid (42.5 mg, 0.34 mmol,
6.0 equiv), DIEA (58.8 mg, 0.46 mmol, 8.0 equiv), DMAP (7 mg, 0.05
mmol, 1.0 equiv) and EDC (32.6 mg, 0.17 mmol, 3.0 equiv) was added.
After the addition was complete, the mixture was stirred at room
temperature for 14 h.
[0162] The mixture was concentrated and purified by pre-HPLC to get
target product as a white solid (2.6 mg), which was confirmed by
NMR and UPLC/MS.
[0163] The examples of Table R were obtained in analogy to Table
Q
TABLE-US-00020 Examples ##STR00215## NQL-023
C.sub.50H.sub.84N.sub.2O.sub.6 MW: 809 [M - H].sup.-: 808
##STR00216## NQL-024 C.sub.54H.sub.94N.sub.4O.sub.10S.sub.2 MW:
1023 [M - H].sup.-: 1022 ##STR00217## NQL-052
C.sub.55H.sub.93NO.sub.10S MW: 960 [M - H].sup.-: 959 ##STR00218##
NQL-053 C.sub.49H.sub.75NO.sub.8S MW: 838 [M - H].sup.-: 837
##STR00219## NQL-054 C.sub.58H.sub.92N.sub.2O.sub.12S.sub.2 MW:
1073 [M - H].sup.-: 1072 ##STR00220## NQL-056
C.sub.41H.sub.61NO.sub.7S MW: 712 [M - H].sup.-: 711
TABLE-US-00021 TABLE S The synthetic route to NQL-052 ##STR00221##
##STR00222## ##STR00223## ##STR00224##
[0164] In a 100 mL, one-necked, round-bottom flask,
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[.alpha.]phenanthren-1'7-yl) pentanoic acid (10.0
g, 25.5 mmol, 1.0 equiv) was dissolved in MeOH (100 mL) at room
temperature. Then PTSA (cat.) was added. After the addition was
complete, the mixture was stirred at 80.degree. C. for 14 h. The
mixture was concentrated and basified with con.NaHCO.sub.3 to
pH>8, then extracted with EA, concentrated under reduced
pressure to get target product (9.3 g) which was confirmed by
NMR.
[0165] In a 100 mL, three-necked, round-bottom flask, PPh.sub.3
(2.6 g, 10.0 mmol, 2.0 equiv) was dissolved in anhydrous THF (100
mL) at -15.degree. C., then DIAD (2.02 g, 10 mmol, 2.0 equiv) was
added under Ar dropwise. After 10 min, methyl
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahy-
dro-1H-cyclopenta[.alpha.]phenanthren-17-yl) pentanoate (2.0 g, 5.0
mmol, 1.0 equiv) dissolved in THF (10 mL) was added dropwise. The
mixture was stirred at room temperature for 30 min. Then
methanesulfonic acid (960 mg, 10.0 mmol, 2.0 equiv) was added.
After the addition was complete, the mixture was stirred at room
temperature for 14 h. The mixture was quenched by the addition of
con.NaHCO.sub.3 to pH>8 and extracted with EA, concentrated
under reduced pressure and purified by silica column chromatography
(PE:EA=1:1) to get target product (0.6 g) which was confirmed by
NMR.
[0166] In a 25 mL, one-necked, round-bottom flask, methyl
(4R)-4-((3S,7S,9S,10S,13R,14S,17R)-7-hydroxy-10,13-dimethyl-3-((methylsul-
fonyl)oxy)hexadecahydro-1H-cyclopenta[.alpha.]phenanthren-17-yl)
pentanoate (1.2 g, 2.5 mmol, 1.0 equiv) was dissolved in DMF (10
mL) at room temperature. Then NaN.sub.3 (325 mg, 5.0 mmol, 2.0
equiv) was added. After the addition was complete, the mixture was
stirred at 80.degree. C. for 1.5 h. The mixture was washed with
water and extracted with EA, concentrated and used directly.
[0167] The crude product was dissolved in MeOH (10 mL) at room
temperature. Then Pd/C (0.5 g) was added and bubbled with a balloon
full of H.sub.2. After the addition was complete, the mixture was
stirred at room temperature for 14 h under H.sub.2. The mixture was
concentrated and purified by chromatography column (DCM: MeOH=5:1)
to get target product as a yellow solid (700 mg), which was
confirmed by NMR.
[0168] In a 10 mL, one-necked, round-bottom flask, methyl
(4R)-4-((3R,7S,9S,10S,13R,14S,17R)-3-amino-7-hydroxy-10,13-dimethylhexade-
cahydro-1H-cyclopenta[.alpha.]phenanthren-17-yl) pentanoate (120.0
mg, 0.3 mmol, 1.0 equiv) was dissolved in DMF (2.0 mL) at room
temperature. Then 2-glutaric acid (39.6 mg, 0.3 mmol, 1.0 equiv),
DIEA (116.0 mg, 0.9 mmol, 3.0 equiv), DMAP (36 mg, 0.3 mmol, 1.0
equiv) and EDC (173 mg, 0.9 mmol, 3.0 equiv) was added. After the
addition was complete, the mixture was stirred at room temperature
for 24 h. The mixture was confirmed by UPLC, concentrated and
purified by chromatography column (DCM: MeOH=20:1) to get target
product (52 mg).
[0169] In a 10 mL, one-necked, round-bottom flask, methyl
(4R)-4-((3S,7R,10S,13R,17R)-7-hydroxy-3-(5-(((3R,7S,10S,13R,17R)-7-hydrox-
y-17-((R)-5-methoxy-5-oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclo-
penta[.alpha.]phenanthren-3-yl)amino)-5-oxopentanamido)-10,13-dimethylhexa-
decahydro-1H-cyclopenta [.alpha.]phenanthren-17-yl) pentanoate
(45.0 mg, 0.05 mmol, 1.0 equiv) was dissolved in THF (1.0 mL)/MeOH
(0.2 mL)/H.sub.2O (1.0 mL) at room temperature. Then LiOH (7.2 mg,
0.17 mmol, 3.0 equiv) was added and stirred at room temperature for
4 h. The mixture was acidified with 1N HCl to pH<3 and extracted
with EA, concentrated and used directly.
[0170] The crude product was dissolved in DMF (1.5 mL) at room
temperature. Then ethane-1,2-diol (2.2 mg, 0.035 mmol, 0.7 equiv),
DIEA (25.8 mg, 0.2 mmol, 4.0 equiv), DMAP (6 mg, 0.05 mmol, 1.0
equiv) and DCC (31.0 mg, 0.15 mmol, 3.0 equiv) was added. After the
addition was complete, the mixture was stirred at room temperature
for 14 h. The mixture was concentrated and purified by pre-HPLC to
get target product as a white solid (3.4 mg), which was confirmed
by NMR.
[0171] The examples of Table T were obtained in analogy to Table
S
TABLE-US-00022 Examples ##STR00225## NQL-058
C.sub.53H.sub.86N.sub.2O.sub.6 MW: 847 [M + H].sup.+: 848
##STR00226## NQL-059 C.sub.55H.sub.88N.sub.2O.sub.8 MW: 905 [M +
H].sup.+: 906
* * * * *